Multiplex detection of vulvovaginal candidiasis, trichomoniasis and bacterial vaginosis

ABSTRACT

Methods and compositions for detection of vulvovaginal candidiasis (VVC), trichomoniasis and bacterial vaginosis (BV) are disclosed herein. In some embodiments, the presence or absence of VVC-associated Candida, Trichomonas vaginalis, and a plurality of BV-related bacteria in a sample is determined using multiplex nucleic acid-based testing methods.

RELATED APPLICATIONS

The present application is a divisional application of U.S. patentapplication Ser. No. 15/567,051 filed on Oct. 16, 2017, which is theU.S. National Phase under 35 U.S.C. § 371 of International PatentApplication No. PCT/US2016/028433 entitled “Multiplex Detection ofVulvovaginal Candidiasis, Trichomoniasis and Bacterial Vaginosis,” filedon Apr. 20, 2016, which claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Application No. 62/152,754, filed on Apr. 24, 2015; and U.S.Provisional Application No. 62/279,220, filed on Jan. 15, 2016. Thecontent of these related applications is herein expressly incorporatedby reference in its entirety.

REFERENCE TO SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledSEQLISTING_GENOM.143WO.TXT, created Apr. 20, 2016, which is 36 Kb insize. The information in the electronic format of the Sequence Listingis incorporated herein by reference in its entirety

BACKGROUND Field

The present disclosure relates to methods and compositions for thedetection of vaginal disorders, for example vulvovaginal candidiasis(VVC), trichomoniasis, and bacterial vaginosis (BV). More specifically,the present disclosure relates to the detection of VVC-associatedCandida species, Trichomonas vaginalis (T. vaginalis) and a plurality ofBV-related bacteria in biological examples, such as vaginal swab samplesfrom women with clinical symptoms of vaginitis and/or vaginosis, bynucleic acid-based test methods.

Description of the Related Art

Candida is a genus of yeast and is the most common cause of fungalinfections worldwide. Many Candida species are found as a harmlesscommensal, part of a normal flora of a host and can be endosymbionts ofhosts including humans. However, in the case of an imbalance or animmunocompromisation of a host, Candida is known to invade and causedisease. Some Candida species, such as C. albicans, C. dubliniensis, C.tropicalis, C. parapsilosis, C. krusei, and C. glabrata, are known to beassociated with vulvovaginal candidiasis (VVC). Trichomonas vaginalis isan anaerobic, flagellated protozoan parasite, which is the causativeagent of trichomoniasis. Bacterial vaginosis (BV) is an infection ofvagina caused by alteration in normal balance of bacteria in the vagina.

To date, standard tests for diagnosing VVC, trichomoniasis, and BV relyon multiple subjective methods that are interpretive methods. Thesetests typically involve microscopic examination of wet mount preparationof patient samples (e.g., vaginal discharge), including observation offungal hyphae or budding yeast for VVC and observation of motiletrichomonads for trichomoniasis. The Nugent Score and Amsel's criteriaare the most commonly used tests for diagnosing BV. The Nugent Score isa Gram stain scoring system by calculated by assessing for the presenceof large Gram-positive rods (Lactobacillus morphotypes), smallGram-variable rods (Gardnerella vaginalis morphotypes), and curvedGram-variable rods (Mobiluncus spp. morphotypes). Amsel's criteriarequires at least three of the four following criteria to be present fora confirmed diagnosis: (1) thin, white, yellow, homogeneous discharge,(2) clue cells on microscopy, (3) pH of vaginal fluid >4.5, and (4)release of a fishy odor on adding alkali-10% potassium hydroxide (KOH)solution. These standard tests can be expensive, labor intensive andtime consuming, for example, Candida needs to be cultured for 48 hourson chromogenic media or up to 7 days on less selective media before adiagnose can be made.

Accordingly, there is a need for developing more efficient and fastermethods for detecting vulvovaginal candidiasis, trichomoniasis andbacterial vaginosis, for example a method allowing detecting of thethree vaginal disorders in a single assay, in order to effectivelydeliver proper treatments to patients.

SUMMARY

Disclosed herein are methods and compositions for detecting vulvovaginalcandidiasis (VVC), trichomoniasis, and/or bacterial vaginosis (BV).

In one aspect, a method to detect a plurality of BV-related bacteria ina biological sample is disclosed, wherein the plurality of BV-relatedbacteria comprises Lactobacillus crispatus, Lactobacillus jensenii,Gardnerella vaginalis, Atopobium vaginae, Megasphaera Type 1, and BVAB2.In some embodiments, the method comprises:

-   -   contacting said biological sample, with a plurality of pairs of        primers, wherein the plurality of pairs of primer comprises:        -   at least one pair of primers capable of hybridizing to the            16S rRNA genes of Lactobacillus crispatus and Lactobacillus            jensenii, wherein each primer in said at least one pair of            primers comprises a sequence of SEQ ID NO: 14 or SEQ ID NO:            15, or a sequence that exhibits at least about 85% identity            to SEQ ID NO: 14 or SEQ ID NO: 15,        -   at least one pair of primers capable of hybridizing to the            16S rRNA gene of BVAB2, wherein each primer in said at least            one pair of primers comprises a sequence of SEQ ID NO: 4 or            SEQ ID NO: 5 or a sequence that exhibits at least about 85%            identity to SEQ ID NO: 4 or SEQ ID NO: 5,        -   at least one pair of primers capable of hybridizing to the            16S rRNA gene of Megasphaera type 1, wherein each primer in            said at least one pair of primers comprises a sequence of            SEQ ID NO: 7 or SEQ ID NO: 8 or a sequence that exhibits at            least about 85% identity to SEQ ID NO: 7 or SEQ ID NO: 8,        -   at least one pair of primers capable of hybridizing to the            vly gene of Gardnerella vaginalis, wherein each primer in            said at least one pair of primers comprises a sequence            selected from the group consisting of SEQ ID NOS: 10-12 or a            sequence that exhibits at least about 85% identity to a            sequence selected from the group consisting of SEQ ID NOS:            10-12, and        -   at least one pair of primers capable of hybridizing to the            16S rRNA gene of Atopobium vaginae, wherein each primer in            said at least one pair of primers comprises a sequence of            SEQ ID NO: 1 or SEQ ID NO: 2, or sequence that exhibits at            least about 85% identity to SEQ ID NO: 1 or SEQ ID NO: 2;    -   generating amplicons of the 16S rRNA sequences of Atopobium        vaginae, BVAB2, Megasphaera type 1, and/or Lactobacillus        crispatus and Lactobacillus jensenii, and/or amplicons of the        vly gene sequence of Gardnerella vaginalis from said biological        sample, if said sample comprises one or more of the BV-related        bacteria; and    -   determining the presence or amount of one or more amplified        products as an indication of the presence of BV-related bacteria        in said biological sample.    -   In some embodiments, the “contacting” step further comprises        contacting said biological sample and said primers with DNA        polymerase, a plurality of free nucleotides comprising adenine,        thymine, cytosine and guanine, and/or a buffer to produce a        reaction mixture. The nucleic acids extracted from the        biological sample may comprise or consist of double stranded        DNA. A reaction mixture may optionally further contain biovalent        cations, monovalent cation potassium ions, one or more        detectably labeled probes, and/or any combination thereof.    -   In some embodiments, the “generating amplicons” step        involves (a) heating the reaction mixture to a first        predetermined temperature for a first predetermined period of        time to separate strands of double stranded DNA present in the        biological sample or in the nucleic acids, (b) cooling the        reaction mixture to a second predetermined temperature for a        second predetermined time under conditions to allow the primers        to hybridize with their complementary sequences and to allow the        DNA polymerase to extend the primers, and (c) repeating        steps (a) and (b) at least 10 to 12 times. In some embodiments,        steps (a) and (b) are repeated at least 15, 20, 22 or 25 times.

In some embodiments, the biological sample is a clinical sample. In someembodiments, the biological sample is collected from the urethra, penis,anus, throat, cervix, or vagina. In some embodiments, the biologicalsample is DNA, RNA or total nucleic acids extracted from a clinicalspecimen.

In some embodiments, the plurality of pairs of primers comprises a firstprimer comprising the sequence of SEQ ID NO: 1, a second primercomprising the sequence of SEQ ID NO: 2, a third primer comprising thesequence of SEQ ID NO: 4, a fourth primer comprising the sequence of SEQID NO: 5, a fifth primer comprising the sequence of SEQ ID NO: 7, asixth primer comprising the sequence of SEQ ID NO: 8, a seventh primercomprising the sequence of SEQ ID NO: 10, an eighth primer comprisingthe sequence of SEQ ID NO: 11, an ninth primer comprising the sequenceof SEQ ID NO: 12, a tenth primer comprising the sequence of SEQ ID NO:14, and an eleventh primer comprising the sequence of SEQ ID NO: 15.

In some embodiments, the pair of primers capable of hybridizing to the16S rRNA genes of Lactobacillus crispatus and Lactobacillus jensenii isSEQ ID NOs: 1 and 2; the pair of primers capable of hybridizing to the16S rRNA gene of BVAB2 is SEQ ID NOs: 4 and 5; the pair of primerscapable of hybridizing to the 16S rRNA gene of Megasphaera type 1 is SEQID NOs: 7 and 8; the pair of primers capable of hybridizing to the vlygene of Gardnerella vaginalis is: a) SEQ ID NOs: 10 and 12, or b) SEQ IDNOs: 11 and 12; and the pair of primers capable of hybridizing to the16S rRNA gene of Atopobium vaginae is SEQ ID NOs: 1 and 2.

In some embodiments, the amplification is carried out using a methodselected from the group consisting of polymerase chain reaction (PCR),ligase chain reaction (LCR), loop-mediated isothermal amplification(LAMP), strand displacement amplification (SDA), replicase-mediatedamplification, Immuno-amplification, nucleic acid sequence basedamplification (NASBA), self-sustained sequence replication (3SR),rolling circle amplification, and transcription-mediated amplification(TMA). For example, the PCR can be real-time PCR. In some embodiments,the PCR is quantitative real-time PCR (QRT-PCR). In some embodiments,each primer comprises exogenous nucleotide sequence which allowspost-amplification manipulation of amplification products without asignificant effect on amplification itself. In some embodiments, eachprimer is flanked by complementary sequences comprising a fluorophore atthe 5′ end, and a fluorescence quencher at the 3′ end.

In some embodiments, determining the presence or amount of one or moreamplified products comprises contacting the amplified products with aplurality of oligonucleotide probes, wherein each of the plurality ofoligonucleotide probes comprises a sequence selected from the groupconsisting of SEQ ID NOs: 3, 6, 9, 13, and 16, or a sequence thatexhibits at least about 85% identity to a sequence selected from thegroup consisting of SEQ ID NOs: 3, 6, 9, 13, and 16. In someembodiments, each of the plurality of oligonucleotide probes comprises,or consists of, a sequence selected from the group consisting of SEQ IDNOs: 3, 6, 9, 13, and 16. In some embodiments, at least one of theplurality of oligonucleotide probes comprises a fluorescence emittermoiety and a fluorescence quencher moiety.

The present disclosure also provides a composition for the detection ofa plurality of BV-related bacteria, wherein the plurality of BV-relatedbacteria comprises Lactobacillus crispatus, Lactobacillus jensenii,Gardnerella vaginalis, Atopobium vaginae, Megasphaera Type 1, and BVAB2.In some embodiments, the composition comprises:

-   -   at least one pair of primers capable of hybridizing to the 16S        rRNA genes of Lactobacillus crispatus and Lactobacillus        jensenii, wherein each primer in said at least one pair of        primers comprises a sequence of SEQ ID NO: 14 or SEQ ID NO: 15,        or a sequence that exhibits at least about 85% identity to SEQ        ID NO: 14 or SEQ ID NO: 15,    -   at least one pair of primers capable of hybridizing to the 16S        rRNA gene of BVAB2, wherein each primer in said at least one        pair of primers comprises a sequence of SEQ ID NO: 4 or SEQ ID        NO: 5 or a sequence that exhibits at least about 85% identity to        SEQ ID NO: 4 or SEQ ID NO: 5,    -   at least one pair of primers capable of hybridizing to the 16S        rRNA gene of Megasphaera type 1, wherein each primer in said at        least one pair of primers comprises a sequence of SEQ ID NO: 7        or SEQ ID NO: 8 or a sequence that exhibits at least about 85%        identity to SEQ ID NO: 7 or SEQ ID NO: 8,    -   at least one pair of primers capable of hybridizing to the vly        gene of Gardnerella vaginalis, wherein each primer in said at        least one pair of primers comprises a sequence selected from the        group consisting of SEQ ID NOS: 10-12 or a sequence that        exhibits at least about 85% identity to a sequence selected from        the group consisting of SEQ ID NOS: 10-12, and    -   at least one pair of primers capable of hybridizing to the 16S        rRNA gene of Atopobium vaginae, wherein each primer in said at        least one pair of primers comprises a sequence of SEQ ID NO: 1        or SEQ ID NO: 2, or sequence that exhibits at least about 85%        identity to SEQ ID NO: 1 or SEQ ID NO: 2.

In some embodiments, the at least one pair of primers capable ofhybridizing to the 16S rRNA genes of Lactobacillus crispatus andLactobacillus jensenii comprises a primer comprising the sequence of SEQID NO: 1 and a primer comprising the sequence of SEQ ID NO: 2; the atleast one pair of primers capable of hybridizing to the 16S rRNA gene ofBVAB2a comprises a primer comprising the sequence of SEQ ID NO: 4 and aprimer comprising the sequence of SEQ ID NO: 5; the at least one pair ofprimers capable of hybridizing to the 16S rRNA gene of Megasphaera type1 comprises a primer comprising the sequence of SEQ ID NO: 7 and aprimer comprising the sequence of SEQ ID NO: 8; the at least one pair ofprimers capable of hybridizing to the vly gene of Gardnerella vaginaliscomprises a primer comprising the sequence of SEQ ID NO: 10 and a primercomprising the sequence of SEQ ID NO: 11; and the at least one pair ofprimers capable of hybridizing to the 16S rRNA gene of Atopobium vaginaecomprises a primer comprising the sequence of SEQ ID NO: 12, a primercomprising the sequence of SEQ ID NO: 14, and a primer comprising thesequence of SEQ ID NO: 15.

The composition can, in some embodiments, further comprises a pluralityof oligonucleotide probes, wherein each of the plurality ofoligonucleotide probes comprises a sequence selected from the groupconsisting of SEQ ID NOs: 3, 6, 9, 13, and 16, or a sequence thatexhibits at least about 85% identity to a sequence selected from thegroup consisting of SEQ ID NOs: 3, 6, 9, 13, and 16. In someembodiments, each of the plurality of oligonucleotide probes comprises,or consists of, a sequence selected from the group consisting of SEQ IDNOs: 3, 6, 9, 13, and 16. In some embodiments, at least one of theplurality of probes comprises a fluorescence emitter moiety and afluorescence quencher moiety.

In another aspect, the present disclosure provides a method to detectvulvovaginal candidiasis (VVC)-associated Candida species andTrichomonas vaginalis in a biological sample, wherein the VVC-associatedCandida species comprises Candida glabrata, Candida albicans, Candidatropicalis, C. dubliniensis, C. parapsilosis, Candida krusei. In someembodiments, the method comprises:

-   -   contacting said biological sample with a plurality of pairs of        primers, wherein the plurality of pairs of primer comprises:        -   at least one pair of primers capable of hybridizing to the            tef1 gene of Candida glabrata, wherein each primer in said            at least one pair of primers comprises a sequence of SEQ ID            NO: 20 or SEQ ID NO: 21 or a sequence that exhibits at least            about 85% identity to SEQ ID NO: 20 or SEQ ID NO: 21;        -   a plurality of primers capable of hybridizing to the tef1            gene of at least one of Candida albicans, Candida            tropicalis, C. dubliniensis, and C. parapsilosis, wherein            each primer in said at least one pair of primers comprises a            sequence of SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 25,            or a sequence that exhibits at least about 85% identity to            SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 25;        -   at least one pair of primers capable of hybridizing to the            tef1 gene of Candida krusei, wherein each primer in said at            least one pair of primers comprises a sequence of SEQ ID NO:            27 or SEQ ID NO: 28, or sequence that exhibits at least            about 85% identity to SEQ ID NO: 27 or SEQ ID NO: 28; and        -   at least one pair of primers capable of hybridizing to the            AP-65 gene of Trichomonas vaginalis, wherein each primer in            said at least one pair of primers comprises a sequence of            SEQ ID NO: 17 or SEQ ID NO: 18, or sequence that exhibits at            least about 85% identity to SEQ ID NO: 17 or SEQ ID NO: 18;            and    -   generating amplicons of the tef1 sequences of the Candida        species and/or amplicons of the AP-65 gene sequence of        Trichomonas vaginalis from said biological sample, if said        sample comprises one or more of the VVC-associated Candida        species and/or Trichomonas vaginalis;    -   determining the presence or amount of one or more amplified        products as an indication of the presence of VVC-associated        Candida species and Trichomonas vaginalis in said biological        sample.    -   In some embodiments, the “contacting” step further comprises        contacting said biological sample and said primers with DNA        polymerase, a plurality of free nucleotides comprising adenine,        thymine, cytosine and guanine, and/or a buffer to produce a        reaction mixture. The nucleic acids extracted from the        biological sample may comprise or consist of double stranded        DNA. A reaction mixture may optionally further contain biovalent        cations, monovalent cation potassium ions, one or more        detectably labeled probes, and/or any combination thereof.    -   In some embodiments, the “generating amplicons” step        involves (a) heating the reaction mixture to a first        predetermined temperature for a first predetermined period of        time to separate strands of double stranded DNA present in the        biological sample or in the nucleic acids, (b) cooling the        reaction mixture to a second predetermined temperature for a        second predetermined time under conditions to allow the primers        to hybridize with their complementary sequences and to allow the        DNA polymerase to extend the primers, and (c) repeating        steps (a) and (b) at least 10 to 12 times. In some embodiments,        steps (a) and (b) are repeated at least 15, 20, 22 or 25 times.

In some embodiments, the biological sample is a clinical sample. In someembodiments, the biological sample is collected from the urethra, penis,anus, throat, cervix, or vagina. In some embodiments, the biologicalsample is DNA, RNA or total nucleic acids extracted from a clinicalspecimen.

In some embodiments, the plurality of pairs of primers comprises a firstprimer comprising the sequence of SEQ ID NO: 20, a second primercomprising the sequence of SEQ ID NO: 21, a third primer comprising thesequence of SEQ ID NO: 23, a fourth primer comprising the sequence ofSEQ ID NO: 24, a fifth primer comprising the sequence of SEQ ID NO: 25,a sixth primer comprising the sequence of SEQ ID NO: 27, a seventhprimer comprising the sequence of SEQ ID NO: 28, an eighth primercomprising the sequence of SEQ ID NO: 17, and an ninth primer comprisingthe sequence of SEQ ID NO: 18.

In some embodiments, the pair of primers capable of hybridizing to thetef1 gene of Candida glabrata is SEQ ID NOs: 20 and 21; the primerscapable of hybridizing to the tef1 gene of at least one of Candidaalbicans, Candida tropicalis, C. dubliniensis, and C. parapsilosis are:a) SEQ ID NOs: 23 and 24, b) SEQ ID NOs: 23 and 35, or c) a combinationthereof, the pair of primers capable of hybridizing to the tef1 gene ofCandida krusei consists of SEQ ID NOs: 27 and 28; and the pair ofprimers capable of hybridizing to the 16S rRNA gene of Trichomonasvaginalis is SEQ ID NOs: 17 and 18.

In some embodiments, the amplification is carried out using a methodselected from the group consisting of polymerase chain reaction (PCR),ligase chain reaction (LCR), loop-mediated isothermal amplification(LAMP), strand displacement amplification (SDA), replicase-mediatedamplification, Immuno-amplification, nucleic acid sequence basedamplification (NASBA), self-sustained sequence replication (3SR),rolling circle amplification, and transcription-mediated amplification(TMA). For example, the PCR can be real-time PCR. In some embodiments,the PCR is quantitative real-time PCR (QRT-PCR).

In some embodiments, each primer comprises exogenous nucleotide sequencewhich allows post-amplification manipulation of amplification productswithout a significant effect on amplification itself. In someembodiments, each primer is flanked by complementary sequencescomprising a fluorophore at the 5′ end, and a fluorescence quencher atthe 3′ end.

In some embodiments, determining the presence or amount of one or moreamplified products comprises contacting the amplified products with aplurality of oligonucleotide probes, wherein each of the plurality ofoligonucleotide probes comprises a sequence selected from the groupconsisting of SEQ ID NOs: 3, 6, 9, 13, and 16, or a sequence thatexhibits at least about 85% identity to a sequence selected from thegroup consisting of SEQ ID NOs: 3, 6, 9, 13, and 16. In someembodiments, each of the plurality of oligonucleotide probes comprises,or consists of, a sequence selected from the group consisting of SEQ IDNOs: 3, 6, 9, 13, and 16.

In some embodiments, at least one of the plurality of oligonucleotideprobes comprises a fluorescence emitter moiety and a fluorescencequencher moiety.

Also disclosed herein is a composition for the detection of vulvovaginalcandidiasis (VVC)-associated Candida species and Trichomonas vaginalisin a biological sample, wherein the VVC-associated Candida speciescomprises Candida glabrata, Candida albicans, Candida tropicalis, C.dubliniensis, C. parapsilosis, Candida krusei. In some embodiments, thecomposition comprises:

-   -   at least one pair of primers capable of hybridizing to the tef1        gene of Candida glabrata, wherein each primer in said at least        one pair of primers comprises a sequence of SEQ ID NO: 20 or SEQ        ID NO: 21 or a sequence that exhibits at least about 85%        identity to SEQ ID NO: 20 or SEQ ID NO: 21;    -   a plurality of primers capable of hybridizing to the tef1 gene        of at least one of Candida albicans, Candida tropicalis, C.        dubliniensis, and C. parapsilosis, wherein each primer in said        at least one pair of primers comprises a sequence of SEQ ID NO:        23, SEQ ID NO: 24, or SEQ ID NO: 25, or a sequence that exhibits        at least about 85% identity to SEQ ID NO: 23, SEQ ID NO: 24, or        SEQ ID NO: 25;    -   at least one pair of primers capable of hybridizing to the tef1        gene of Candida krusei, wherein each primer in said at least one        pair of primers comprises a sequence of SEQ ID NO: 27 or SEQ ID        NO: 28, or sequence that exhibits at least about 85% identity to        SEQ ID NO: 27 or SEQ ID NO: 28; and    -   at least one pair of primers capable of hybridizing to the AP-65        gene of Trichomonas vaginalis, wherein each primer in said at        least one pair of primers comprises a sequence of SEQ ID NO: 17        or SEQ ID NO: 18, or sequence that exhibits at least about 85%        identity to SEQ ID NO: 17 or SEQ ID NO: 18.

In some embodiments, the at least one pair of primers capable ofhybridizing to the tef1 gene of Candida glabrata comprises a primercomprising the sequence of SEQ ID NO: 20 and a primer comprising thesequence of SEQ ID NO: 21; the plurality of primers capable ofhybridizing to the tef1 gene of at least one of Candida albicans,Candida tropicalis, C. dubliniensis, and C. parapsilosis comprises aprimer comprising the sequence of SEQ ID NO: 23, a primer comprising thesequence of SEQ ID NO: 24, and a primer comprising the sequence of SEQID NO: 25; the at least one pair of primers capable of hybridizing tothe tef1 gene of Candida krusei comprises a primer comprising thesequence of SEQ ID NO: 27 and a primer comprising the sequence of SEQ IDNO: 28; and the at least one pair of primers capable of hybridizing tothe AP-65 gene of Trichomonas vaginalis comprises a primer comprisingthe sequence of SEQ ID NO: 17 and a primer comprising the sequence ofSEQ ID NO: 18.

In some embodiments, the composition can further comprises a pluralityof oligonucleotide probes, wherein each of the plurality ofoligonucleotide probes comprises a sequence selected from the groupconsisting of SEQ ID NOs: 22, 26, 29, and 19, or a sequence thatexhibits at least about 85% identity to a sequence selected from thegroup consisting of SEQ ID NOs: 22, 26, 29, and 19. In some embodiments,each of the plurality of oligonucleotide probes comprises, or consistsof a sequence selected from the group consisting of SEQ ID NOs: 22, 26,29, and 19. In some embodiments, at least one of the plurality of probescomprises a fluorescence emitter moiety and a fluorescence quenchermoiety.

In one aspect, the present disclosure provides oligonucleotide probes orprimers up to about 100 nucleotides in length which are capable ofhybridizing to vaginolysin gene (vly) of Gardnerella vaginalis, whereinsaid probe or primer comprises a sequence selected from the groupconsisting of SEQ ID NOs: 10-13, or sequence that exhibits at leastabout 85% identity to a sequence selected from the group consisting ofSEQ ID NOs: 10-13. In some embodiments, the probe or primer consists ofa sequence selected from the group consisting of SEQ ID NOs: 10-13, orsequence that exhibits at least about 85% identity to a sequenceselected from the group consisting of SEQ ID NOs: 10-13. In someembodiments, the probe or primer comprises a sequence selected from thegroup consisting of SEQ ID NOs: 10-13. In some embodiments, the probe orprimer consists of a sequence selected from the group consisting of SEQID NOs: 10-13.

DETAILED DESCRIPTION

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described inany way. All literature and similar materials cited in this applicationincluding, but not limited to, patents, patent applications, articles,books, treatises, and internet web pages, regardless of the format ofsuch literature and similar materials, are expressly incorporated byreference in their entirety for any purpose. In the event that one ormore of the incorporated literature and similar materials defines oruses a term in such a way that it contradicts that term's definition inthis application, this application controls. While the present teachingsare described in conjunction with various embodiments, it is notintended that the present teachings be limited to such embodiments. Onthe contrary, the present teachings encompass various alternatives,modifications, and equivalents, as will be appreciated by those of skillin the art.

Provided herein are methods and compositions for the detection ofvulvovaginal candidiasis (VVC), trichomoniasis, and bacterial vaginosis(BV). For example, primers and probes that can bind to specific genes ofCandida species associated with VVC, Trichomonas vaginalis (T.vaginalis) and BV-related bacteria are provided to determine thepresence or absence of the VVC-associated Candida species, T. vaginalisand BV-related bacteria in a sample, such as a biological sample. Insome embodiments, multiplex nucleic acid amplification can be performedto allow the detection of VVC-associated Candida species, T. vaginalisand BV-related bacteria in a single assay.

Definitions

As used herein, a “nucleic acid” refers to a polymeric compoundcomprising nucleosides or nucleoside analogs which have nitrogenousheterocyclic bases, or base analogs, linked together by nucleic acidbackbone linkages (e.g., phosphodiester bonds) to form a polynucleotide.Non-limiting examples of nucleic acid include RNA, DNA, and analogsthereof. The nucleic acid backbone can include a variety of linkages,for example, one or more of sugar-phosphodiester linkages,peptide-nucleic acid bonds, phosphorothioate or methylphosphonatelinkages or mixtures of such linkages in a single oligonucleotide. Sugarmoieties in the nucleic acid can be either ribose or deoxyribose, orsimilar compounds with known substitutions. Conventional nitrogenousbases (e.g., A, G, C, T, U), known base analogs (e.g., inosine),derivatives of purine or pyrimidine bases and “abasic” residues (i.e.,no nitrogenous base for one or more backbone positions) are included inthe term nucleic acid. That is, a nucleic acid can include onlyconventional sugars, bases and linkages found in RNA and DNA, or includeboth conventional components and substitutions (e.g., conventional basesand analogs linked via a methoxy backbone, or conventional bases and oneor more base analogs linked via an RNA or DNA backbone).

As used herein, the term “isolate nucleic acids” refers to thepurification of nucleic acids from one or more cellular components. Oneof skill in the art will appreciate that samples processed to “isolatenucleic acids” therefrom can include components and impurities otherthan nucleic acids. Samples that comprise isolated nucleic acids can beprepared from specimens using any acceptable method known in the art.For example, cells can be lysed using known lysis agents, and nucleicacids can be purified or partially purified from other cellularcomponents. Suitable reagents and protocols for DNA and RNA extractionscan be found in, for example, U.S. Patent Application Publication Nos.US 2010-0009351, and US 2009-0131650, respectively (each of which isincorporated herein by reference in its entirety). In nucleic acidtesting (e.g., amplification and hybridization methods discussed infurther detail below), the extracted nucleic acid solution can be addeddirectly to a reagents (e.g., either in liquid, bound to a substrate, inlyophilized form, or the like, as discussed in further detail below),required to perform a test according to the embodiments disclosedherein.

As used herein, “template” refers to all or part of a polynucleotidecontaining at least one target nucleotide sequence.

As used herein, a “primer” refers to a polynucleotide that can serve toinitiate a nucleic acid chain extension reaction. The length of a primercan vary, for example, from about 5 to about 100 nucleotides, from about10 to about 50 nucleotides, from about 15 to about 40 nucleotides, orfrom about 20 to about 30 nucleotides. The length of a primer can beabout 10 nucleotides, about 20 nucleotides, about 25 nucleotides, about30 nucleotides, about 35 nucleotides, about 40 nucleotides, about 50nucleotides, about 75 nucleotides, about 100 nucleotides, or a rangebetween any two of these values. In some embodiments, the primer has alength of 10 to about 50 nucleotides, i.e., 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or morenucleotides. In some embodiments, the primer has a length of 18 to 32nucleotides.

As used herein, a “probe” refers to an polynucleotide that canhybridizes (e.g., specifically) to a target sequence in a nucleic acid,under conditions that allow hybridization, thereby allowing detection ofthe target sequence or amplified nucleic acid. A probe's “target”generally refers to a sequence within or a subset of an amplifiednucleic acid sequence which hybridizes specifically to at least aportion of a probe oligomer by standard hydrogen bonding (i.e., basepairing). A probe may comprise target-specific sequences and othersequences that contribute to three-dimensional conformation of theprobe. Sequences are “sufficiently complementary” if they allow stablehybridization in appropriate hybridization conditions of a probeoligomer to a target sequence that is not completely complementary tothe probe's target-specific sequence. The length of a probe can vary,for example, from about 5 to about 100 nucleotides, from about 10 toabout 50 nucleotides, from about 15 to about 40 nucleotides, or fromabout 20 to about 30 nucleotides. The length of a probe can be about 10nucleotides, about 20 nucleotides, about 25 nucleotides, about 30nucleotides, about 35 nucleotides, about 40 nucleotides, about 50nucleotides, about 100 nucleotides, or a range between any two of thesevalues. In some embodiments, the probe has a length of 10 to about 50nucleotides. For example, the primers and or probes can be at least 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, or more nucleotides. In some embodiments, the probe canbe non-sequence specific.

Preferably, the primers and/or probes can be between 8 and 45nucleotides in length. For example, the primers and or probes can be atleast 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, or more nucleotides in length. The primer and probe can bemodified to contain additional nucleotides at the 5′ or the 3′ terminus,or both. One of skill in the art will appreciate that additional basesto the 3′ terminus of amplification primers (not necessarily probes) aregenerally complementary to the template sequence. The primer and probesequences can also be modified to remove nucleotides at the 5′ or the 3′terminus. One of skill in the art will appreciate that in order tofunction for amplification, the primers or probes will be of a minimumlength and annealing temperature as disclosed herein.

Primers and probes can bind to their targets at an annealingtemperature, which is a temperature less than the melting temperature(T_(m)). As used herein, “T_(m)” and “melting temperature” areinterchangeable terms which refer to the temperature at which 50% of apopulation of double-stranded polynucleotide molecules becomesdissociated into single strands. The formulae for calculating the T_(m)of polynucleotides are well known in the art. For example, the T_(m) maybe calculated by the following equation: T_(m)=69.3+0.41×(G+C) %−6−50/L,wherein L is the length of the probe in nucleotides. The T_(m) of ahybrid polynucleotide may also be estimated using a formula adopted fromhybridization assays in 1 M salt, and commonly used for calculatingT_(m) for PCR primers: [(number of A+T)×2° C.+(number of G+C)×4° C.].See, e.g., C. R. Newton et al. PCR, 2nd ed., Springer-Verlag (New York:1997), p. 24 (incorporated by reference in its entirety, herein). Othermore sophisticated computations exist in the art, which take structuralas well as sequence characteristics into account for the calculation ofT_(m). The melting temperature of an oligonucleotide can depend oncomplementarity between the oligonucleotide primer or probe and thebinding sequence, and on salt conditions. In some embodiments, anoligonucleotide primer or probe provided herein has a T_(m) of less thanabout 90° C. in 50 mM KCl, 10 mM Tris-HCl buffer, for example about 89°C., 88, 87, 86, 85, 84, 83, 82, 81, 80 79, 78, 77, 76, 75, 74, 73, 72,71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54,53, 52, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39° C., or less,including ranges between any two of the listed values.

In some embodiments, the primers disclosed herein, e.g., amplificationprimers, can be provided as an amplification primer pair, e.g.,comprising a forward primer and a reverse primer (first amplificationprimer and second amplification primer). Preferably, the forward andreverse primers have T_(m)'s that do not differ by more than 10° C.,e.g., that differ by less than 10° C., less than 9° C., less than 8° C.,less than 7° C., less than 6° C., less than 5° C., less than 4° C., lessthan 3° C., less than 2° C., or less than 1° C.

The primer and probe sequences may be modified by having nucleotidesubstitutions (relative to the target sequence) within theoligonucleotide sequence, provided that the oligonucleotide containsenough complementarity to hybridize specifically to the target nucleicacid sequence. In this manner, at least 1, 2, 3, 4, or up to about 5nucleotides can be substituted. As used herein, the term “complementary”refers to sequence complementarity between regions of two polynucleotidestrands or between two regions of the same polynucleotide strand. Afirst region of a polynucleotide is complementary to a second region ofthe same or a different polynucleotide if, when the two regions arearranged in an antiparallel fashion, at least one nucleotide of thefirst region is capable of base pairing with a base of the secondregion. Therefore, it is not required for two complementarypolynucleotides to base pair at every nucleotide position. “Fullycomplementary” refers to a first polynucleotide that is 100% or “fully”complementary to a second polynucleotide and thus forms a base pair atevery nucleotide position. “Partially complementary” also refers to afirst polynucleotide that is not 100% complementary (e.g., 90%, or 80%or 70% complementary) and contains mismatched nucleotides at one or morenucleotide positions. In some embodiments, an oligonucleotide includes auniversal base.

As used herein, an “exogenous nucleotide sequence” refers to a sequenceintroduced by primers or probes used for amplification, such thatamplification products will contain exogenous nucleotide sequence andtarget nucleotide sequence in an arrangement not found in the originaltemplate from which the target nucleotide sequence was copied.

As used herein, “sequence identity” or “percent identical” as applied tonucleic acid molecules is the percentage of nucleic acid residues in acandidate nucleic acid molecule sequence that are identical with asubject nucleic acid molecule sequence, after aligning the sequences toachieve the maximum percent identity, and not considering any nucleicacid residue substitutions as part of the sequence identity. Nucleicacid sequence identity can be determined using any method known in theart, for example CLUSTALW, T-COFFEE, BLASTN.

As used herein, the term “sufficiently complementary” refers to acontiguous nucleic acid base sequence that is capable of hybridizing toanother base sequence by hydrogen bonding between a series ofcomplementary bases. Complementary base sequences can be complementaryat each position in the oligomer sequence by using standard base pairing(e.g., G:C, A:T or A:U) or can contain one or more residues that are notcomplementary (including abasic positions), but in which the entirecomplementary base sequence is capable of specifically hybridizing withanother base sequence in appropriate hybridization conditions.Contiguous bases can be at least about 80%, at least about 85%, at leastabout 90%, at least about 95%, at least about 99%, or 100% complementaryto a sequence to which an oligomer is intended to hybridize.Substantially complementary sequences can refer to sequences ranging inpercent identity from 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89,88, 87, 86, 85, 84, 83, 82, 81, 80, 75, 70 or less, or any number inbetween, compared to the reference sequence. A skilled artisan canreadily choose appropriate hybridization conditions which can bepredicted based on base sequence composition, or be determined by usingroutine testing (see e.g., Green and Sambrook, Molecular Cloning, ALaboratory Manual, 4th ed. (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 2012)).

As used herein, the term “multiplex PCR” refers to a type of PCR wheremore than one set of primers is included in a reaction allowing onesingle target, or two or more different targets to be amplified in asingle reaction tube. The multiplex PCR can be, for example, a real-timePCR.

Oligonucleotides and Compositions Containing Thereof

As described herein, nucleic acid amplifications can be performed todetermine the presence, absence and/or level of Candida species, T.vaginalis, and/or BV-related bacteria in a sample. Some Candida speciesare known to be associated with VVC, including but not limited to C.albicans, C. dubliniensis, C. tropicalis, C. parapsilosis, C. krusei,and C. glabrata. Many bacteria are also known to be related to BV,including but not limited to, Lactobacillus spp. (for exampleLactobacillus crispatus (L. crispatus) and Lactobacillus jensenii (L.jensenii)), Gardnerella vaginalis (G. vaginalis), Atopobium vaginae,Megasphaera Type 1 (Megasphaera-1), and BVAB-2. In some embodiments, thepresence, absence and/or level of VVC-associated Candida species, T.vaginalis, and BV-related bacteria is determined by detecting one ormore target genes of each of the target organisms using methods known inthe art, such as DNA amplifications. In some embodiments, a multiplexPCR can be performed to detect the presence, absence or level for eachof the target Candida species, T. vaginalis, and/or BV-related bacteria.In some embodiments, a multiplex PCR is performed to detect thepresence, absence and/or level for each of target VVC-associated Candidaspecies, T. vaginalis, L. crispatus, L. jensenii, G. vaginalis,Atopobium vaginae, Megasphaera Type 1, and BVAB-2. In some embodiments,the VVC-associated Candida species are C. albicans, C. dubliniensis, C.tropicalis, C. parapsilosis, C. krusei, and C. glabrata.

Each of the target VVC-associated Candida species, T. vaginalis, andBV-related bacteria can be detected using separate channels in DNAamplifications. In some cases, it can be desirable to use a singlefluorescence channel for detecting the presence, absence, and/or levelof two or more of the VVC-associated Candida species, T. vaginalis, andBV-related bacteria. For example, a single fluorescence channel can beused to detect the presence, absence, and/or level of two BV-relatedbacteria (e.g., BVAB-2 and Megasphaera-1). Such combination may, in someembodiments, reduce the amount of reagent needed to conduct theexperiment as well as provide an accurate qualitative metric upon whicha BV determination can be assessed. Without being bound any particulartheory, it is believed that the use of combined markers may increase thesensitivity and specificity of the assay. In some embodiments, separatefluorescence channels are used to detect the presence, absence and/orlevel of each of Lactobacillus spp. (for example L. crispatus and L.jensenii), G. vaginalis, and Atopobium vaginae, and a singlefluorescence channel is used to detect the presence, absence, and/orlevel of BVAB-2 and Megasphaera-1.

Oligonucleotides (for example amplification primers and probes) that arecapable of specifically hybridizing (e.g., under standard nucleic acidamplification conditions, e.g., standard PCR conditions, and/orstringent hybridization conditions) to a target gene region, orcomplement thereof, in VVC-associated Candida species, T. vaginalis, L.crispatus, L. jensenii, G. vaginalis, Atopobium vaginae, MegasphaeraType 1 (Megasphaera-1), and BVAB-2 are provided. Amplification of thetarget gene region of an organism in a sample (e.g., a vaginal swabsample) can, in some embodiments, be indicative of the presence,absence, and/or level of the organism in the sample.

The target gene region can vary. In some embodiments, oligonucleotides(e.g., amplification primers and probes) that are capable ofspecifically hybridizing (e.g., under standard nucleic acidamplification conditions, e.g., standard PCR conditions, and/orstringent hybridization conditions) to a gene region encoding 16Sribosomal RNA (16S rRNA) in an organism is provided. In someembodiments, the organism is Atopobium vaginae. In some embodiments, theorganism is BVAB2. In some embodiments, the organism is Megasphaeratype 1. In some embodiments, the organism is L. crispatus. In someembodiments, the microorganism is L. jensenii. In some embodiments, 16SrRNA gene is used as the target gene for the DNA amplification to detectthe presence, absence and/or level of Atopobium vaginae, BVAB-2,Megasphaera type 1, L. crispatus, and/or L. jensenii in the sample.Examples of oligonucleotides capable of specifically hybridizing to the16S rRNA gene region in BVAB-2 include, but are not limited, SEQ ID NOs:4-6 as provided in Table 1 and sequences that exhibits at least about85% identity to a sequence selected from the group consisting of SEQ IDNOs: 4-6. Examples of oligonucleotides capable of specificallyhybridizing to the 16S rRNA gene region in Megasphaera type 1 include,but are not limited, SEQ ID NOs: 7-9 as provided in Table 1 andsequences that exhibits at least about 85% identity to a sequenceselected from the group consisting of SEQ ID NOs: 7-9. In someembodiments, primers and probes that can specifically bind to the 16SrRNA gene region of Atopobium vaginae are used in detection of thepresence, absence and/or level of Atopobium vaginae in a biologicalsample. Examples of oligonucleotides capable of specifically hybridizingto the 16S rRNA gene region in Atopobium vaginae include, but are notlimited, SEQ ID NOs: 1-3 as provided in Table 1 and sequences thatexhibits at least about 85% identity to a sequence selected from thegroup consisting of SEQ ID NOs: 1-3. Examples of oligonucleotidescapable of specifically hybridizing to the 16S rRNA gene region in L.crispatus and L. jensenii include, but are not limited, SEQ ID NOs:14-16 as provided in Table 1 and sequences that exhibits at least about85% identity to a sequence selected from the group consisting of SEQ IDNOs: 14-16.

Toxin vaginolysin (VLY) is the main virulence factor of G. vaginalis,encoded by the gene vly. VLY belongs to the cholesterol dependentcytolysins, a family of pore forming toxins, and is known to disruptplasma membranes causing cell lysis and are thought to play a key rolein the virulence of G. vaginalis. In some embodiments, oligonucleotides(e.g., amplification primers and probes) that are capable ofspecifically hybridizing (e.g., under standard nucleic acidamplification conditions, e.g., standard PCR conditions, and/orstringent hybridization conditions) to a gene region encoding vly in G.vaginalis are provided. In some embodiments, vaginolysin (vly) gene isused as the target gene for the DNA amplification to detect thepresence, absence and/or level of G. vaginalis in the sample. In someembodiments, primers and probes that can specifically bind to the vlygene region of G. vaginalis are used in detection of the presence,absence and/or level of G. vaginalis in a biological sample. Examples ofoligonucleotides capable of specifically hybridizing to the vly generegion in G. vaginalis include, but are not limited, SEQ ID NOs: 10-13as provided in Table 1 and sequences that exhibits at least about 85%identity to a sequence selected from the group consisting of SEQ ID NOs:10-13.

Protein AP65 is a 65 KDa protein by the parasitic organism T. vaginalis,which upon iron repletion acts as a surface adhesin that mediatescytoadherence of the parasite to vaginal epithelial cells. In someembodiments disclosed herein, oligonucleotides (e.g., amplificationprimers and probes) that are capable of specifically hybridizing (e.g.,under standard nucleic acid amplification conditions, e.g., standard PCRconditions, and/or stringent hybridization conditions) to a gene regionencoding AP65 in T. vaginalis are provided. In some embodiments, AP65gene is used as the target gene for the DNA amplification to detect thepresence, absence and/or level of T. vaginalis in the sample. Examplesof oligonucleotides capable of specifically hybridizing to the AP65 generegion in T. vaginalis include, but are not limited, SEQ ID NOs: 17-19as provided in Table 1 and sequences that exhibits at least about 85%identity to a sequence selected from the group consisting of SEQ ID NOs:17-19.

The elongation factor 1 alpha (tef1) gene found in Candida speciesencodes for protein synthesis factor EF, which is involved in thetranslational process during protein synthesis. As known in the art,tef1 gene is often referred to as tef1 gene or tuf gene as well. In someembodiments disclosed herein, oligonucleotides (e.g., amplificationprimers and probes) that are capable of specifically hybridizing (e.g.,under standard nucleic acid amplification conditions, e.g., standard PCRconditions, and/or stringent hybridization conditions) to a gene regionencoding tef in Candida species are provided. In some embodiments, tef1gene is used as the target gene for the DNA amplification to detect thepresence, absence and/or level of VVC-associated Candida species in thesample. In some embodiments, the VVC-associated Candida speciescomprises C. albicans, C. dubliniensis, C. tropicalis, C. parapsilosis,C. krusei, and C. glabrata. In some embodiments, the VVC-associatedCandida species is Candida krusei. In some embodiments, theVVC-associated Candida species is Candida glabrata. In some embodiments,the VVC-associated Candida species is C. albicans, C. dubliniensis, C.tropicalis, C. parapsilosis, or a combination thereof. Examples ofoligonucleotides capable of specifically hybridizing to the tef1 generegion in C. glabrata include, but are not limited, SEQ ID NOs: 20-22 asprovided in Table 1 and sequences that exhibits at least about 85%identity to a sequence selected from the group consisting of SEQ ID NOs:20-22. Examples of oligonucleotides capable of specifically hybridizingto the tef1 gene region in C. albicans, C. dubliniensis, C. tropicalis,and C. parapsilosis include, but are not limited, SEQ ID NOs: 23-26 asprovided in Table 1 and sequences that exhibits at least about 85%identity to a sequence selected from the group consisting of SEQ ID NOs:23-26. Examples of oligonucleotides capable of specifically hybridizingto the tef1 gene region in C. krusei include, but are not limited, SEQID NOs: 27-29 as provided in Table 1 and sequences that exhibits atleast about 85% identity to a sequence selected from the groupconsisting of SEQ ID NOs: 27-29.

TABLE 1 Primer and probes for detection of VVC-associatedCandida species, T. vaginalis and BV-related species Primer/ TargetTargeted Probe Primer/Probe Organism gene Name Sequences (5′-3′)Atopobium 16S rRNA MenAv248fw CCCTATCCGCTCCTGATACC (SEQ ID NO: 1)vaginae 16S rRNA MenAv334rv CCAAATATCTGCGCATTTCA (SEQ ID NO: 2) 16S rRNAMCF-Av-T4 TCCCCTACCAGACTCAAGCCTGC (SEQ ID NO: 3)(5′ fluorophore: FAM, 3′ fluorophore: BHQ1) BVAB2 16S rRNA 585F_BVAB2GCGGCTAGATAAGTGTGATGTTT (SEQ ID NO: 4) 16S rRNA 666R_BVAB2CTCTCCAGCACTCAAGCTAAA (SEQ ID NO: 5) 16S rRNA BVAB2_613_641CAAGGCTTAACCTTGGGGTTCATTACAA (SEQ ID NO: 6)(5′ fluorophore: CFO, 3′ fluorophore: BHQ1) Megasphaera 16S rRNA456F_MegaE GATGCCAACAGTATCCGTCCG (SEQ ID NO: 7) type 1 16S rRNA667R_MegaE CCTCTCCGACACTCAAGTTCGA (SEQ ID NO: 8) 16S rRNA Mega485-506-TTACCGTAAGAGAAAGCCACGG (SEQ ID NO: 9)(5′ fluorophore: CFO, 3′ fluorophore: BHQ1) Gardnerella vly GVvlyfw2GCCAACGATGATCGCGTAT (SEQ ID NO: 10) vaginalis vly GVvlyfw2amodGCCAATAATGACCGCGTAT (SEQ ID NO: 11) vly GVvlyrv1AGCCGTTCACTGCGGAAGT (SEQ ID NO: 12) vly MCF-Gv-T3ACAGCACTTTCGCCGCC (SEQ ID NO: 13)(5′ fluorophore: Quasar670, 3′ fluorophore: BHQ2) Lactobacillus 16S rRNAMCF-Lj_Lc-F8 TTAAAAGGCGGCGTAAGC (SEQ ID NO: 14) crispatus and 16S rRNAMCF-Lsp-R6 GCCAGTTACTACCTCTATC (SEQ ID NO: 15) Lactobacillus 16S rRNAMCF-Lsp-T11 AAGTCTGATGGAGCAACGCC (SEQ ID NO: 16) jensenii(5′ fluorophore: ROX, 3′ fluorophore: BHQ2) Trichomonas AP-65 TV.MAX.FP1GAAGATTCTGGCAAGATCAAGGA (SEQ ID NO: 17) vaginalis AP-65 TV.MAX.RP1ACGACAATGCAGCGGATGT (SEQ ID NO: 18) AP-65 TV.MAX.D1-TATCCTCCGCAACTACCCACGCCA (SEQ ID NO: 19)(5′ fluorophore: FAM, 3′ fluorophore: BHQ1) Candida tef1 SiT-Cgla-F8CGAACAATTGACTGAAGGTTTG (SEQ ID NO: 20) glabrata tef1 RT-Cgla-R7CGGACTTCAAGAACTTTGGAGA (SEQ ID NO: 21) tef1 RT-Cgla-T7CTTGTAAGTTCGAAGAATTGTTGGA (SEQ ID NO: 22)(5′ fluorophore: CFO, 3′ fluorophore: BHQ1) Candida tef1 RT-Ca-Cd-Ct-F1CCACCAAAGGGTTGTGAC (SEQ ID NO: 23) genus* tef1 RT-Ca-Ct-R3CAGCATCACCGGATTTGAC (SEQ ID NO: 24) tef1 RT-Cpar-R6CGGACTTGATGAATTTTGGTTCA (SEQ ID NO: 25) tef1 RT-Ca-Cd-T3TGCTTGTAAATTCGACACTTTGGTTG (SEQ ID NO: 26)(5′ fluorophore: ROX, 3′ fluorophore: BHQ2) Candida tef1 RT-Ckru-F7GCAGCTTCCTTCAATGCTCAA (SEQ ID NO: 27) krusei tef1 SiT-Ckru-R10aATCACCAGACTTGACAG (SEQ ID NO: 28) tef1 RT-Ckru-T9CATGTAAGTTCGACGAATTAATCGA (SEQ ID NO: 29)(5′ fluorophore: Quasar670, 3′ fluorophore: BHQ2) Controls DrosScaff2DrosScaff2-LP GGCATGGAGGTTGTCCCATTTGTG (SEQ ID NO: 30) DrosScaff2DrosScaff2-UP GGATCTAGCCGTGTGCCCGCT (SEQ ID NO: 31) DrosScaff2 Sign-T1TTGATGCCTCTTCACATTGCTCCACCTTTCCT (SEQ ID NO: 32)(5′ fluorophore: Quasar705, 3′ fluorophore: BHQ3) * C. albicans, C.dubliniensis, C. tropicalis, or C. parapsilosis

Also provided herein are oligonucleotides (for example amplificationprimers or probes) containing 1, 2, 3, 4 or more mismatches or universalnucleotides relative to SEQ ID NOs: 1-32 or the complement thereof,including oligonucleotides that are at least 80% identical (for exampleat least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) to SEQ ID NOs: 1-32or the complement thereof. In some embodiments, the oligonucleotidecomprises a sequence selected from SEQ ID NO: 1-32. In some embodiments,the oligonucleotide comprises a sequence that is at least about 85%identical to a sequence selected from SEQ ID NO: 1-32. In someembodiments, the oligonucleotide consists of a sequence selected fromSEQ ID NO: 1-32. In some embodiments, the oligonucleotide consists of asequence that is at least about 85% identical or at least about 95%identical to a sequence selected from SEQ ID NO: 1-32.

The nucleic acids provided herein can be in various forms. For example,in some embodiments, the nucleic acids are dissolved (either alone or incombination with various other nucleic acids) in solution, for examplebuffer. In some embodiments, nucleic acids are provided, either alone orin combination with other isolated nucleic acids, as a salt. In someembodiments, nucleic acids are provided in a lyophilized form that canbe reconstituted. For example, in some embodiments, the isolated nucleicacids disclosed herein can be provided in a lyophilized pellet alone, orin a lyophilized pellet with other isolated nucleic acids. In someembodiments, nucleic acids are provided affixed to a solid substance,such as a bead, a membrane, or the like. In some embodiments, nucleicacids are provided in a host cell, for example a cell line carrying aplasmid, or a cell line carrying a stably integrated sequence.

Also disclosed herein are compositions, reaction mixtures, and kits thatcomprise the oligonucleotides (e.g., amplification primers and/orprobes) that are capable of specifically hybridizing to the sequence ofthe 16S rRNA gene of Atopobium vaginae, BVAB-2, Megasphaera type 1, L.crispatus, and/or L. jensenii, or a complement thereof. In someembodiments, the composition, reaction mixture, and kit comprise one ormore pairs of amplification primers capable of specifically hybridizingto the sequence of 16S rRNA sequence, or a complement thereof, ofAtopobium vaginae. In some embodiments, the primer comprises a sequenceof SEQ ID NO: 1 or 2. In some embodiments, the primer comprises asequence that is at least about 85% identical, at least about 90%, or atleast about 95% identical to a sequence of SEQ ID NO: 1 or 2. In someembodiments, the primer consists of a sequence of SEQ ID NO: 1 or 2. Insome embodiments, the primer consists of a sequence that is at leastabout 85% identical, at least about 90% identical, or at least about 95%identical to a sequence of SEQ ID NO: 1 or 2. In some embodiments, thecomposition, reaction mixture, and kit comprise one or more probescapable of specifically hybridizing to the sequence of 16S rRNA gene, orcomplement thereof, of Atopobium vaginae. In some embodiments, the probecomprises a sequence of SEQ ID NO: 3. In some embodiments, the probecomprises a sequence that is at least about 85% identical, at leastabout 90% identical, or at least about 95% identical to a sequence ofSEQ ID NO: 3. In some embodiments, the probe consists of a sequence ofSEQ ID NO: 3. In some embodiments, the probe consists of a sequence thatis at least about 85% identical, at least about 90% identical, or atleast about 95% identical to a sequence of SEQ ID NO: 3.

In some embodiments, the composition, reaction mixture, and kit compriseone or more pairs of amplification primers capable of specificallyhybridizing to the sequence of 16S rRNA sequence, or a complementthereof, of BVAB-2. In some embodiments, the primer comprises a sequenceof SEQ ID NO: 4 or 5. In some embodiments, the primer comprises asequence that is at least about 85% identical, at least about 90%, or atleast about 95% identical to a sequence of SEQ ID NO: 4 or 5. In someembodiments, the primer consists of a sequence of SEQ ID NO: 4 or 5. Insome embodiments, the primer consists of a sequence that is at leastabout 85% identical, at least about 90% identical, or at least about 95%identical to a sequence of SEQ ID NO: 4 or 5. In some embodiments, thecomposition, reaction mixture, and kit comprise one or more probescapable of specifically hybridizing to the sequence of 16S rRNA gene, orcomplement thereof, of BVAB-2. In some embodiments, the probe comprisesa sequence of SEQ ID NO: 6. In some embodiments, the probe comprises asequence that is at least about 85% identical, at least about 90%identical, or at least about 95% identical to a sequence of SEQ ID NO:6. In some embodiments, the probe consists of a sequence of SEQ ID NO:6. In some embodiments, the probe consists of a sequence that is atleast about 85% identical, at least about 90% identical, or at leastabout 95% identical to a sequence of SEQ ID NO: 6.

In some embodiments, the composition, reaction mixture, and kit compriseone or more pairs of amplification primers capable of specificallyhybridizing to the sequence of 16S rRNA sequence, or a complementthereof, of Megasphaera type 1. In some embodiments, the primercomprises a sequence of SEQ ID NO: 7 or 8. In some embodiments, theprimer comprises a sequence that is at least about 85% identical, atleast about 90%, or at least about 95% identical to a sequence of SEQ IDNO: 7 or 8. In some embodiments, the primer consists of a sequence ofSEQ ID NO: 7 or 8. In some embodiments, the primer consists of asequence that is at least about 85% identical, at least about 90%identical, or at least about 95% identical to a sequence of SEQ ID NO: 7or 8. In some embodiments, the composition, reaction mixture, and kitcomprise one or more probes capable of specifically hybridizing to thesequence of 16S rRNA gene, or complement thereof, of Megasphaera type 1.In some embodiments, the probe comprises a sequence of SEQ ID NO: 9. Insome embodiments, the probe comprises a sequence that is at least about85% identical, at least about 90% identical, or at least about 95%identical to a sequence of SEQ ID NO: 9. In some embodiments, the probeconsists of a sequence of SEQ ID NO: 9. In some embodiments, the probeconsists of a sequence that is at least about 85% identical, at leastabout 90% identical, or at least about 95% identical to a sequence ofSEQ ID NO: 9.

In some embodiments, the composition, reaction mixture, and kit compriseone or more pairs of amplification primers capable of specificallyhybridizing to the sequence of 16S rRNA sequence, or a complementthereof, of L. crispatus and/or L. jensenii. In some embodiments, theprimer comprises a sequence of SEQ ID NO: 14 or 15. In some embodiments,the primer comprises a sequence that is at least about 85% identical, atleast about 90%, or at least about 95% identical to a sequence of SEQ IDNO: 14 or 15. In some embodiments, the primer consists of a sequence ofSEQ ID NO: 14 or 15. In some embodiments, the primer consists of asequence that is at least about 85% identical, at least about 90%identical, or at least about 95% identical to a sequence of SEQ ID NO:14 or 15. In some embodiments, the composition, reaction mixture, andkit comprise one or more probes capable of specifically hybridizing tothe sequence of 16S rRNA gene, or complement thereof, of L. crispatusand/or L. jensenii. In some embodiments, the probe comprises a sequenceof SEQ ID NO: 16. In some embodiments, the probe comprises a sequencethat is at least about 85% identical, at least about 90% identical, orat least about 95% identical to a sequence of SEQ ID NO: 16. In someembodiments, the probe consists of a sequence of SEQ ID NO: 16. In someembodiments, the probe consists of a sequence that is at least about 85%identical, at least about 90% identical, or at least about 95% identicalto a sequence of SEQ ID NO: 16.

Compositions, reaction mixtures, and kits that that comprise theoligonucleotides (e.g., amplification primers and/or probes) that arecapable of specifically hybridizing to the sequence of vly gene of G.vaginalis, or a complement thereof, are also provided. In someembodiments, the composition, reaction mixture, and kit comprise one ormore pairs of amplification primers capable of specifically hybridizingto the sequence of vly gene sequence of G. vaginalis, or a complementthereof. In some embodiments, the primer comprises a sequence of SEQ IDNO: 10, 11, or 12. In some embodiments, the primer comprises a sequencethat is at least about 85% identical, at least about 90%, or at leastabout 95% identical to a sequence of SEQ ID NO: 10, 11 or 12. In someembodiments, the primer consists of a sequence of SEQ ID NO: 10, 11 or12. In some embodiments, the primer consists of a sequence that is atleast about 85% identical, at least about 90% identical, or at leastabout 95% identical to a sequence of SEQ ID NO: 10, 11 or 12. In someembodiments, the composition, reaction mixture, and kit comprise one ormore probes capable of specifically hybridizing to the sequence of vlygene of G. vaginalis, or complement thereof. In some embodiments, theprobe comprises a sequence of SEQ ID NO: 13. In some embodiments, theprobe comprises a sequence that is at least about 85% identical, atleast about 90% identical, or at least about 95% identical to a sequenceof SEQ ID NO: 13. In some embodiments, the probe consists of a sequenceof SEQ ID 13. In some embodiments, the probe consists of a sequence thatis at least about 85% identical, at least about 90% identical, or atleast about 95% identical to a sequence of SEQ ID NO: 13.

Compositions, reaction mixture, and kits that that comprise theoligonucleotides (e.g., amplification primers and/or probes) that arecapable of specifically hybridizing to the sequence of the AP-65 gene ofT. vaginalis, or a complement thereof, are provided. In someembodiments, the composition, reaction mixture, and kit comprise one ormore pairs of amplification primers capable of specifically hybridizingto the sequence of the AP-65 gene sequence of T. vaginalis, or acomplement thereof. In some embodiments, the primer comprises a sequenceof SEQ ID NO: 17 or 18. In some embodiments, the primer comprises asequence that is at least about 85% identical, at least about 90%, or atleast about 95% identical to a sequence of SEQ ID NO: 17 or 18. In someembodiments, the primer consists of a sequence of SEQ ID NO: 17 or 18.In some embodiments, the primer consists of a sequence that is at leastabout 85% identical, at least about 90% identical, or at least about 95%identical to a sequence of SEQ ID NO: 17 or 18. In some embodiments, thecomposition, reaction mixture, and kit comprise one or more probescapable of specifically hybridizing to the sequence of AP-65 gene of T.vaginalis, or complement thereof. In some embodiments, the probecomprises a sequence of SEQ ID NO: 19. In some embodiments, the probecomprises a sequence that is at least about 85% identical, at leastabout 90% identical, or at least about 95% identical to a sequence ofSEQ ID NO: 19. In some embodiments, the probe consists of a sequence ofSEQ ID 19. In some embodiments, the probe consists of a sequence that isat least about 85% identical, at least about 90% identical, or at leastabout 95% identical to a sequence of SEQ ID NO: 19.

Compositions, reaction mixtures, and kits that comprise theoligonucleotides (e.g., amplification primers and/or probes) that arecapable of specifically hybridizing to the sequence of tef1 gene of oneor more Candida species, or complement thereof, are provided. In someembodiments, the composition, reaction mixture, and kit comprise one ormore pairs of amplification primers capable of specifically hybridizingto the sequence of tef1 gene sequence of Candida glabrata, or complementthereof. In some embodiments, the primer comprises a sequence of SEQ IDNO: 20 or 21. In some embodiments, the primer comprises a sequence thatis at least about 85% identical, at least about 90%, or at least about95% identical to a sequence of SEQ ID NO: 20 or 21. In some embodiments,the primer consists of a sequence of SEQ ID NO: 20 or 21. In someembodiments, the primer consists of a sequence that is at least about85% identical, at least about 90% identical, or at least about 95%identical to a sequence of SEQ ID NO: 20 or 21. In some embodiments, thecomposition, reaction mixture, and kit comprise one or more probescapable of specifically hybridizing to the sequence of tef1 gene Candidaglabrata, or complement thereof. In some embodiments, the probecomprises a sequence of SEQ ID NO: 22, 26 or 29. In some embodiments,the probe comprises a sequence that is at least about 85% identical, atleast about 90% identical, or at least about 95% identical to a sequenceof SEQ ID NO: 22. In some embodiments, the probe consists of a sequenceof SEQ ID NO: 22. In some embodiments, the probe consists of a sequencethat is at least about 85% identical, at least about 90% identical, orat least about 95% identical to a sequence of SEQ ID NO: 22.

In some embodiments, the composition, reaction mixture, and kit compriseone or more pairs of amplification primers capable of specificallyhybridizing to the sequence of tef1 gene sequence of one or more Candidaspecies, or complement thereof, wherein the Candida species comprises C.albicans, C. dubliniensis, C. tropicalis, and C. parapsilosis. In someembodiments, the primer comprises a sequence of SEQ ID NO: 20, 21, 23,24, 25, 27, or 28. In some embodiments, the primer comprises a sequencethat is at least about 85% identical, at least about 90%, or at leastabout 95% identical to a sequence of SEQ ID NO: 23, 24 or 25. In someembodiments, the primer consists of a sequence of SEQ ID NO: 23, 24 or25 In some embodiments, the primer consists of a sequence that is atleast about 85% identical, at least about 90% identical, or at leastabout 95% identical to a sequence of SEQ ID NO: 23, 24 or 25. In someembodiments, the composition, reaction mixture, and kit comprise one ormore probes capable of specifically hybridizing to the sequence of tef1gene of one or more Candida species, or complement thereof, wherein theCandida species comprises C. albicans, C. dubliniensis, C. tropicalis,and C. parapsi. In some embodiments, the probe comprises a sequence ofSEQ ID NO: 26. In some embodiments, the probe comprises a sequence thatis at least about 85% identical, at least about 90% identical, or atleast about 95% identical to a sequence of SEQ ID NO: 26. In someembodiments, the probe consists of a sequence of SEQ ID NO: 26. In someembodiments, the probe consists of a sequence that is at least about 85%identical, at least about 90% identical, or at least about 95% identicalto a sequence of SEQ ID NO: 26.

In some embodiments, the composition, reaction mixture, and kit compriseone or more pairs of amplification primers capable of specificallyhybridizing to the sequence of tef1 gene sequence of Candida krusei, orcomplement thereof. In some embodiments, the primer comprises a sequenceof SEQ ID NO: 27 or 28. In some embodiments, the primer comprises asequence that is at least about 85% identical, at least about 90%, or atleast about 95% identical to a sequence of SEQ ID NO: 27 or 28. In someembodiments, the primer consists of a sequence of SEQ ID NO: 27 or 28.In some embodiments, the primer consists of a sequence that is at leastabout 85% identical, at least about 90% identical, or at least about 95%identical to a sequence of SEQ ID NO: 27 or 28. In some embodiments, thecomposition, reaction mixture, and kit comprise one or more probescapable of specifically hybridizing to the sequence of tef1 gene ofCandida krusei, or complement thereof. In some embodiments, the probecomprises a sequence of SEQ ID NO: 29. In some embodiments, the probecomprises a sequence that is at least about 85% identical, at leastabout 90% identical, or at least about 95% identical to a sequence ofSEQ ID NO: 29. In some embodiments, the probe consists of a sequence ofSEQ ID NO: 29. In some embodiments, the probe consists of a sequencethat is at least about 85% identical, at least about 90% identical, orat least about 95% identical to a sequence of SEQ ID NO: 29.

Oligonucleotide probes can, in some embodiments, include a detectablemoiety. For example, the oligonucleotide probes disclosed herein cancomprise a radioactive label. Non-limiting examples of radioactivelabels include ³H, ¹⁴C, ³²P, and ³⁵S. In some embodiments,oligonucleotide probes can include one or more non-radioactivedetectable markers or moieties, including but not limited to ligands,fluorophores, chemiluminescent agents, enzymes, and antibodies. Otherdetectable markers for use with probes, which can enable an increase insensitivity of the method of the invention, include biotin andradio-nucleotides. It will become evident to the person of ordinaryskill that the choice of a particular label dictates the manner in whichit is bound to the probe. For example, oligonucleotide probes labeledwith one or more dyes, such that upon hybridization to a templatenucleic acid, a detectable change in fluorescence is generated. Whilenon-specific dyes may be desirable for some applications,sequence-specific probes can provide more accurate measurements ofamplification. One configuration of sequence-specific probe can includeone end of the probe tethered to a fluorophore, and the other end of theprobe tethered to a quencher. When the probe is unhybridized, it canmaintain a stem-loop configuration, in which the fluorophore is quenchedby the quencher, thus preventing the fluorophore from fluorescing. Whenthe probe is hybridized to a template nucleic sequence, it islinearized, distancing the fluorophore from the quencher, and thuspermitting the fluorophore to fluoresce. Another configuration ofsequence-specific probe can include a first probe tethered to a firstfluorophore of a FRET pair, and a second probe tethered to a secondfluorophore of a FRET pair. The first probe and second probe can beconfigured to hybridize to sequences of an amplicon that are withinsufficient proximity to permit energy transfer by FRET when the firstprobe and second probe are hybridized to the same amplicon.

In some embodiments, the sequence specific probe comprises anoligonucleotide as disclosed herein conjugated to a fluorophore. In someembodiments, the probe is conjugated to two or more fluorophores.Examples of fluorophores include: xanthene dyes, e.g., fluorescein andrhodamine dyes, such as fluorescein isothiocyanate (FITC),2-[ethylamino)-3-(ethylimino)-2-7-dimethyl-3H-xanthen-9-yl]benzoic acidethyl ester monohydrochloride (R6G)(emits a response radiation in thewavelength that ranges from about 500 to 560 nm),1,1,3,3,3′,3′-Hexamethylindodicarbocyanine iodide (HIDC) (emits aresponse radiation in the wavelength that ranged from about 600 to 660nm), 6-carboxyfluorescein (commonly known by the abbreviations FAM andF), 6-carboxy-2′,4′,7′,4,7-hexachlorofluorescein (HEX),6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein (JOE or J),N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA or T),6-carboxy-X-rhodamine (ROX or R), 5-carboxyrhodamine-6G (R6G5 or G5),6-carboxyrhodamine-6G (R6G6 or G6), and rhodamine 110; cyanine dyes,e.g. Cy3, Cy5 and Cy7 dyes; coumarins, e.g., umbelliferone; benzimidedyes, e.g. Hoechst 33258; phenanthridine dyes, e.g. Texas Red; ethidiumdyes; acridine dyes; carbazole dyes; phenoxazine dyes; porphyrin dyes;polymethine dyes, e.g. cyanine dyes such as Cy3 (emits a responseradiation in the wavelength that ranges from about 540 to 580 nm), Cy5(emits a response radiation in the wavelength that ranges from about 640to 680 nm), etc; BODIPY dyes and quinoline dyes. Specific fluorophoresof interest include: Pyrene, Coumarin, Diethylaminocoumarin, FAM,Fluorescein Chlorotriazinyl, Fluorescein, R110, Eosin, JOE, R6G, HIDC,Tetramethylrhodamine, TAMRA, Lissamine, ROX, Napthofluorescein, TexasRed, Napthofluorescein, Cy3, and Cy5, CAL fluor orange, and the like.

In some embodiments, the probe is conjugated to a quencher. A quenchercan absorb electromagnetic radiation and dissipate it as heat, thusremaining dark. Example quenchers include Dabcyl, NFQ's, such as BHQ-1or BHQ-2 (Biosearch), IOWA BLACK FQ (IDT), and IOWA BLACK RQ (IDT). Insome embodiments, the quencher is selected to pair with a fluorophore soas to absorb electromagnetic radiation emitted by the fluorophore.Fluorophore/quencher pairs useful in the compositions and methodsdisclosed herein are well-known in the art, and can be found, e.g.,described in Marras, “Selection of Fluorophore and Quencher Pairs forFluorescent Nucleic Acid Hybridization Probes” available atwww.molecular-beacons.org/download/marras,mmb06%28335%293.pdf.

In some embodiments, a fluorophore is attached to a first end of theprobe, and a quencher is attached to a second end of the probe.Attachment can include covalent bonding, and can optionally include atleast one linker molecule positioned between the probe and thefluorophore or quencher. In some embodiments, a fluorophore is attachedto a 5′ end of a probe, and a quencher is attached to a 3′ end of aprobe. In some embodiments, a fluorophore is attached to a 3′ end of aprobe, and a quencher is attached to a 5′ end of a probe. Examples ofprobes that can be used in quantitative nucleic acid amplificationinclude molecular beacons, SCORPION™ probes (Sigma), TAQMAN™ probes(Life Technologies) and the like. Other nucleic acid detectiontechnologies that are useful in the embodiments disclosed hereininclude, but are not limited to nanoparticle probe technology (See,Elghanian, et al. (1997) Science 277:1078-1081.) and Amplifluor probetechnology (See, U.S. Pat. Nos. 5,866,366; 6,090,592; 6,117,635; and6,117,986).

Some embodiments provide a composition for the detection of a pluralityof BV-related bacteria, wherein the composition comprises: primerscapable of hybridizing to the 16S rRNA genes of L. crispatus and/or L.jensenii, wherein each primer comprises a sequence of SEQ ID NO: 14 orSEQ ID NO: 15, or a sequence that exhibits at least about 85% identityto SEQ ID NO: 14 or SEQ ID NO: 15; primers capable of hybridizing to the16S rRNA gene of BVAB2, wherein each primer comprises a sequence of SEQID NO: 4 or SEQ ID NO: 5 or a sequence that exhibits at least about 85%identity to SEQ ID NO: 4 or SEQ ID NO: 5; primers capable of hybridizingto the 16S rRNA gene of Megasphaera type 1, wherein each primercomprises a sequence of SEQ ID NO: 7 or SEQ ID NO: 8 or a sequence thatexhibits at least about 85% identity to SEQ ID NO: 7 or SEQ ID NO: 8;primers capable of hybridizing to the vly gene of G. vaginalis, whereineach primer comprises a sequence selected from the group consisting ofSEQ ID NOS: 10-12 or a sequence that exhibits at least about 85%identity to a sequence selected from the group consisting of SEQ ID NOS:10-12, and primers capable of hybridizing to the 16S rRNA gene ofAtopobium vaginae, wherein each primer comprises a sequence of SEQ IDNO: 1 or SEQ ID NO: 2, or sequence that exhibits at least about 85%identity to SEQ ID NO: 1 or SEQ ID NO: 2.

In some embodiments, the primers capable of hybridizing to the 16S rRNAgenes of L. crispatus and/or L. jensenii comprise, or consist of, aprimer comprising the sequence of SEQ ID NO: 1 and a primer comprisingthe sequence of SEQ ID NO: 2; the primers capable of hybridizing to the16S rRNA gene of BVAB2 comprise, or consist of, a primer comprising thesequence of SEQ ID NO: 4 and a primer comprising the sequence of SEQ IDNO: 5; the primers capable of hybridizing to the 16S rRNA gene ofMegasphaera type 1 comprise, consists of, a primer comprising thesequence of SEQ ID NO: 7 and a primer comprising the sequence of SEQ IDNO: 8; the primers capable of hybridizing to the vly gene of G.vaginalis comprise, or consist of, a primer comprising the sequence ofSEQ ID NO: 10 and a primer comprising the sequence of SEQ ID NO: 11; andthe primers capable of hybridizing to the 16S rRNA gene of Atopobiumvaginae comprise, or consist of a primer comprising the sequence of SEQID NO: 12, a primer comprising the sequence of SEQ ID NO: 14, and aprimer comprising the sequence of SEQ ID NO: 15.

The composition can further comprise a plurality of oligonucleotideprobes, wherein each of the plurality of oligonucleotide probescomprises a sequence selected from the group consisting of SEQ ID NOs:3, 6, 9, 13, and 16, or a sequence that exhibits at least about 85%identity to a sequence selected from the group consisting of SEQ ID NOs:3, 6, 9, 13, and 16. In some embodiments, each of the plurality ofoligonucleotide probes comprises, or consists of, a sequence selectedfrom the group consisting of SEQ ID NOs: 3, 6, 9, 13, and 16.

Some embodiments disclosed herein provide a composition for thedetection of VVC-associated Candida species and T. vaginalis in abiological sample, wherein the composition comprises: primers capable ofhybridizing to the tef1 gene of Ca. glabrata, wherein each primercomprises a sequence of SEQ ID NO: 20 or SEQ ID NO: 21 or a sequencethat exhibits at least about 85% identity to SEQ ID NO: 20 or SEQ ID NO:21; primers capable of hybridizing to the tef1 gene of at least one ofC. albicans, C. tropicalis, C. dubliniensis, and C. parapsilosis,wherein each primer comprises a sequence of SEQ ID NO: 23, SEQ ID NO:24, or SEQ ID NO: 25, or a sequence that exhibits at least about 85%identity to SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 25; primerscapable of hybridizing to the tef1 gene of C. krusei, wherein eachprimer comprises a sequence of SEQ ID NO: 27 or SEQ ID NO: 28, orsequence that exhibits at least about 85% identity to SEQ ID NO: 27 orSEQ ID NO: 28; and primers capable of hybridizing to the AP-65 gene ofT. vaginalis, wherein each primer comprises a sequence of SEQ ID NO: 17or SEQ ID NO: 18, or sequence that exhibits at least about 85% identityto SEQ ID NO: 17 or SEQ ID NO: 18.

In some embodiments, the primers capable of hybridizing to the tef1 geneof C. glabrata comprise, or consist of, a primer comprising the sequenceof SEQ ID NO: 20 and a primer comprising the sequence of SEQ ID NO: 21;the primers capable of hybridizing to the tef1 gene of at least one ofC. albicans, C. tropicalis, C. dubliniensis, and C. parapsilosiscomprise, or consist of, a primer comprising the sequence of SEQ ID NO:23, a primer comprising the sequence of SEQ ID NO: 24, and a primercomprising the sequence of SEQ ID NO: 25; the primers capable ofhybridizing to the tef1 gene of C. krusei comprise, or consist of, aprimer comprising the sequence of SEQ ID NO: 27 and a primer comprisingthe sequence of SEQ ID NO: 28; and the primers capable of hybridizing tothe AP-65 gene of T. vaginalis comprise, or consist of a primercomprising the sequence of SEQ ID NO: 17 and a primer comprising thesequence of SEQ ID NO: 18.

The composition can, in some embodiments, further comprises a pluralityof oligonucleotide probes, wherein each of the plurality ofoligonucleotide probes comprises a sequence selected from the groupconsisting of SEQ ID NOs: 22, 26, 29, and 19, or a sequence thatexhibits at least about 85% identity to a sequence selected from thegroup consisting of SEQ ID NOs: 22, 26, 29, and 19. In some embodiments,each of the plurality of oligonucleotide probes comprises, or consistsof, a sequence selected from the group consisting of SEQ ID NOs: 22, 26,29, and 19.

Any probes described herein can comprise a fluorescence emitter moietyand a fluorescence quencher moiety.

As disclosed herein, a reaction mixture can comprise one or more of theprimers disclosed herein, one or more of the probes disclosed herein(e.g., the fluorophore-containing probes), or any combination thereof.In some embodiments, the reaction mixture comprises one or more of theprimer and/or probe-containing composition disclosed herein. Thereaction mixture can also comprise various additional components.Examples of the additional components in the reaction mixture include,but are not limited to, template DNA, DNA polymerase (e.g., Taq DNApolymerase), deoxynucleotides (dNTPs), buffer solution, biovalentcations, monovalent cation potassium ions, and any combination thereof.In some embodiments, the reaction mixture is a master mix for real-timePCR.

Samples

The methods and compositions disclosed herein are suitable for detectingvaginal disorders, such as VVC, trichomoniasis and BV, in a wide varietyof samples. As used herein, a “sample” refers to any type of material ofbiological origin taken from one or more number of subjects that aresuspected of suffering from VVC, trichomoniasis, and/or BV. The samplecan comprise, for example, fluid, tissue or cell. The sample cancomprise a biological material taken directly from a subject, orcultured call or tissues, or any fraction or products produced from orderived from biological materials. A sample can be purified, partiallypurified, unpurified, enriched, or amplified.

The sample can be a biological sample, for example a clinical sample. Insome embodiments, the sample is taken from a biological source, such asvagina, urethra, penis, anus, throat, cervix, fermentation broths, cellcultures, and the like. The sample can comprise, for example, fluid andcells from vagina. The biological sample can be used (i) directly asobtained from the subject or source, or (ii) following a pre-treatmentto modify the character of the sample. Thus, the test sample can bepre-treated prior to use, for example, by disrupting cells or viralparticles, preparing liquids from solid materials, diluting viscousfluids, filtering liquids, concentrating liquids, inactivatinginterfering components, adding reagents, purifying nucleic acids, andthe like. Accordingly, a “biological sample” as used herein includesnucleic acids (DNA, RNA or total nucleic acids) extracted from aclinical or biological specimen. Sample preparation can also includeusing a solution that contains buffers, salts, detergents, and/or thelike which are used to prepare the sample for analysis. In someembodiments, the sample is processed before molecular testing. In someembodiments, the sample is analyzed directly, and is not pre-processedprior to testing. The sample can be, for example, a vaginal sample, suchas a single vaginal swab sample. In some embodiments, the sample is avaginal swab sample from a female with clinical symptoms of vaginitisand/or vaginosis.

Vaginal or urine samples are often infected with multiple organisms. Thedisclosed primers and probes are tolerant to mixed infections of thevaginal or urine matrix.

In some embodiments, a sample to be tested is processed prior toperforming the methods disclosed herein. For example, in someembodiments, the sample can be isolated, concentrated, or subjected tovarious other processing steps prior to performing the methods disclosedherein. For example, in some embodiments, the sample can be processed toisolate nucleic acids from the sample prior to contacting the samplewith the oligonucleotides, as disclosed herein. In some embodiments, themethods disclosed herein are performed on the sample without culturingthe sample in vitro. In some embodiments, the methods disclosed hereinare performed on the sample without isolating nucleic acids from thesample prior to contacting the sample with oligonucleotides as disclosedherein.

Sample Extraction

In typical sample extractions, cells are lysed by mechanical shearingwith glass beads as described in U.S. Pat. No. 7,494,771, incorporatedby reference in its entirety herein, to lyse the target organisms. Asdisclosed in WO03/008636, such a generic method of cell lysis isefficient for a wide variety of target organisms and specimen matrices.There are also other less universal lysis methods that are designedspecifically to target a certain species or group of organisms, or whichexploit specific enzymatic or chemical activities. For example, ACPenzyme is commonly used to lyse of Gram-positive organisms (Ezaki etal., J. Clin. Microbiol., 16(5):844-846 (1982); Paule et al., J. Mol.Diagn., 6(3):191-196 (2004); U.S. Pat. No. 3,649,454; all incorporatedby reference in their entirety herein) and mycobacteria (U.S. Pat. No.5,185,242, incorporated by reference in its entirety) but is generallyconsidered to be less efficacious with respect to lysis of Gram-negativespecies such as E. coli and Pseudomonas aeruginosa (U.S. Pat. No.3,649,454, incorporated by reference in its entirety).

Inventors of the present disclosure was surprised to find that neitherACP nor Proteinase K can efficiently lyse Candida cells walls, andlyticase described in patent U.S. Pat. No. 3,716,452 (incorporated byreference in its entirety) can effectively lyse cell walls of Candidaspecies. Cell lysis can be performed under various temperatures, forexample between 18° C. to 75° C., for example, 37° C. and 50° C. It isadvantageous to lyse the cells at 37° C. to achieve higher lysisefficiency as compared to 50° C. (LND490E38). In some embodiments,lyticase is used to lyse Candida species, including but not limited toC. albicans, C. krusei, C. parapsilosis, C. tropicalis, and C. glabrata.The time required to achieve desired lysis efficiency for the sample isnot particularly limited. In some embodiments, it requires about 10minute to achieve desired lysis efficiency of the sample.

Nucleic Acid Testing

The methods described herein can include, for example, nucleic acidtesting. For example, the test can include testing for target nucleicacid sequences in a sample. Various forms of nucleic acid testing can beused in the embodiments disclosed herein, including but not limited to,testing that involves nucleic acid amplification.

As used herein, nucleic acid amplification refers to any known procedurefor obtaining multiple copies of a target nucleic acid sequence or itscomplement or fragments thereof, using sequence-specific methods.Examples of known amplification methods include, but are not limited to,polymerase chain reaction (PCR), ligase chain reaction (LCR),loop-mediated isothermal amplification (LAMP), strand displacementamplification (SDA) (e.g., multiple displacement amplification (MDA)),replicase-mediated amplification, immuno-amplification, nucleic acidsequence based amplification (NASBA), self-sustained sequencereplication (3SR), rolling circle amplification, andtranscription-mediated amplification (TMA). See, e.g., Mullis, “Processfor Amplifying, Detecting, and/or Cloning Nucleic Acid Sequences,” U.S.Pat. No. 4,683,195; Walker, “Strand Displacement Amplification,” U.S.Pat. No. 5,455,166; Dean et al, “Multiple displacement amplification,”U.S. Pat. No. 6,977,148; Notomi et al., “Process for SynthesizingNucleic Acid,” U.S. Pat. No. 6,410,278; Landegren et al. U.S. Pat. No.4,988,617 “Method of detecting a nucleotide change in nucleic acids”;Birkenmeyer, “Amplification of Target Nucleic Acids Using Gap FillingLigase Chain Reaction,” U.S. Pat. No. 5,427,930; Cashman,“Blocked-Polymerase Polynucleotide Immunoassay Method and Kit,” U.S.Pat. No. 5,849,478; Kacian et al., “Nucleic Acid Sequence AmplificationMethods,” U.S. Pat. No. 5,399,491; Malek et al., “Enhanced Nucleic AcidAmplification Process,” U.S. Pat. No. 5,130,238; Lizardi et al.,BioTechnology, 6:1197 (1988); Lizardi et al., U.S. Pat. No. 5,854,033“Rolling circle replication reporter systems.” In some embodiments, twoor more of the aforementioned nucleic acid amplification methods can beperformed, for example sequentially.

For example, LCR amplification uses at least four separateoligonucleotides to amplify a target and its complementary strand byusing multiple cycles of hybridization, ligation, and denaturation (EPPatent No. 0 320 308). SDA amplifies by using a primer that contains arecognition site for a restriction endonuclease which nicks one strandof a hemimodified DNA duplex that includes the target sequence, followedby amplification in a series of primer extension and strand displacementsteps (U.S. Pat. No. 5,422,252 to Walker et al.).

PCR is a method well-known in the art for amplification of nucleicacids. PCR involves amplification of a target sequence using two or moreextendable sequence-specific oligonucleotide primers that flank thetarget sequence. The nucleic acid containing the target sequence ofinterest is subjected to a program of multiple rounds of thermal cycling(denaturation, annealing and extension) in the presence of the primers,a thermostable DNA polymerase (e.g., Taq polymerase) and various dNTPs,resulting in amplification of the target sequence. PCR uses multiplerounds of primer extension reactions in which complementary strands of adefined region of a DNA molecule are simultaneously synthesized by athermostable DNA polymerase. At the end of each cycle, each newlysynthesized DNA molecule acts as a template for the next cycle. Duringrepeated rounds of these reactions, the number of newly synthesized DNAstrands increases exponentially such that after 20 to 30 reactioncycles, the initial template DNA will have been replicated severalthousand-fold or million-fold. Methods for carrying out different typesand modes of PCR are thoroughly described in the literature, for examplein “PCR Primer: A Laboratory Manual” Dieffenbach and Dveksler, eds. ColdSpring Harbor Laboratory Press, 1995, and by Mullis et al. in patents(e.g., U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159) and scientificpublications (e.g. Mullis et al. 1987, Methods in Enzymology,155:335-350) where the contents of each reference are herebyincorporated by reference in their entireties.

PCR can generate double-stranded amplification products suitable forpost-amplification processing. If desired, amplification products can bedetected by visualization with agarose gel electrophoresis, by an enzymeimmunoassay format using probe-based colorimetric detection, byfluorescence emission technology, or by other detection means known toone of skill in the art.

A wide variety of PCR methods have been described in many sources, forexample, Ausubel et al. (eds.), Current Protocols in Molecular Biology,Section 15, John Wiley & Sons, Inc., New York (1994). Examples of PCRmethod include, but not limited to, Real-Time PCR, End-Point PCR,Amplified fragment length polymorphism PCR (AFLP-PCR), Alu-PCR,Asymmetric PCR, Colony PCR, DD-PCR, Degenerate PCR, Hot-start PCR, Insitu PCR, Inverse PCR Long-PCR, Multiplex PCR, Nested PCR, PCR-ELISA,PCR-RFLP, PCR-single strand conformation polymorphism (PCR-SSCP),quantitative competitive PCR (QC-PCR), rapid amplification of cDNAends-PCR (RACE-PCR), Random Amplification of Polymorphic DNA-PCR(RAPD-PCR), Real-Time PCR, Repetitive extragenic palindromic-PCR(Rep-PCR), reverse transcriptase PCR (RT-PCR), TAIL-PCR, Touchdown PCRand Vectorette PCR.

Real-time PCR, also called quantitative real time polymerase chainreaction (QRT-PCR), can be used to simultaneously quantify and amplify aspecific part of a given nucleic acid molecule. It can be used todetermine whether a specific sequence is present in the sample; and ifit is present, the number of copies of the sequence that are present.The term “real-time” refers to periodic monitoring during PCR. Certainsystems such as the ABI 7700 and 7900HT Sequence Detection Systems(Applied Biosystems, Foster City, Calif.) conduct monitoring during eachthermal cycle at a pre-determined or user-defined point. Real-timeanalysis of PCR with fluorescence resonance energy transfer (FRET)probes measures fluorescent dye signal changes from cycle-to-cycle,preferably minus any internal control signals. The real-time procedurefollows the general pattern of PCR, but the nucleic acid is quantifiedafter each round of amplification. Two examples of method ofquantification are the use of fluorescent dyes (e.g., SYBRGreen) thatintercalate into double-stranded DNA, and modified DNA oligonucleotideprobes that fluoresce when hybridized with a complementary DNA.Intercalating agents have a relatively low fluorescence when unbound,and a relatively high fluorescence upon binding to double-strandednucleic acids. As such, intercalating agents can be used to monitor theaccumulation of double strained nucleic acids during a nucleic acidamplification reaction. Examples of such non-specific dyes useful in theembodiments disclosed herein include intercalating agents such as SYBRGreen I (Molecular Probes), propidium iodide, ethidium bromide, and thelike.

Vaginal samples are often infected with multiple organisms. Thedisclosed primers and probes are tolerant to mixed infections of thevaginal matrix. Because of the specific target sequences, primers andprobes, the methods and compositions disclosed herein can be used todetect the presence/absence or level of VVC-associated Candida species,T. vaginalis, and/or BV-related bacteria in a sample with highsensitivity, specificity and accuracy.

The primers disclosed herein can be paired with additional PCR systemsusing a uniform chemistry and thermal PCR profile to provide a panel ofassays for the detection of vaginal organisms, to improve overall assaysensitivity and robustness.

In some embodiments, multiplex PCR is performed to amplify and detect,e.g., by direct or indirect means, the presence or absence ofVVC-associated Candida species, T. vaginalis, and BV-related bacteria toallow diagnose of VVC, Trichomoniasis and BV using one test. In themultiplex PCR, the presence or absence of VVC-associated Candida speciescan be determined by amplifying and detecting the presence or absence oftef1 gene of C. albicans, C. dubliniensis, C. tropicalis, C.parapsilosis, C. krusei, and C. glabrata; the presence or absence of T.vaginalis can be determined by amplifying and detecting the presence orabsence of AP-65 gene of T. vaginalis; the presence or absence ofBV-related bacteria, including L. crispatus, L. jensenii, G. vaginalis,Atopobium vaginae, Megasphaera Type 1, and BVAB-2, can be determined byamplifying and detecting the presence or absence of 16S rRNA gene ofAtopobium vaginae, BVAB-2, Megasphaera Type 1, and the presence orabsence of vly gene of G. vaginalis.

Accordingly, some embodiments for the detection and/or identification ofVVC-associated Candida species, T. vaginalis, and BV-related bacteria ina sample include the steps of providing a test sample; and contactingthe sample with oligonucleotide primers that can specifically hybridizeand amplify (1) tef1 genes of C. albicans, C. dubliniensis, C.tropicalis, C. parapsilosis, C. krusei, and C. glabrata, (2) AP-65 geneof T. vaginalis, (3) 16S rRNA genes of Atopobium vaginae, BVAB-2,Megasphaera Type 1, and (4) vly gene of G. vaginalis, andoligonucleotide probes that can specifically hybridizes to (1) tef1 generegions of C. albicans, C. dubliniensis, C. tropicalis, C. parapsilosis,C. krusei, and C. glabrata, (2) AP-65 gene region of T. vaginalis, (3)16S rRNA gene regions of Atopobium vaginae, BVAB-2, Megasphaera Type 1,and (4) vly gene region of G. vaginalis under standard nucleic acidamplification conditions and/or stringent hybridization conditions. Asdescribed herein, the sample can be contacted with all of the primersand probes at once, or can be contacted with some of the primers andprobes first and subsequently contacted by the remainder of the primersand probes. In some embodiments, the sample is contacted with theprimers that can specifically hybridize and amplify (1) tef1 genes of C.albicans, C. dubliniensis, C. tropicalis, C. parapsilosis, C. krusei,and C. glabrata, and (2) AP-65 gene of Trichomonas vaginalis, and theprobes that can specifically hybridizes to (1) tef1 gene regions of C.albicans, C. dubliniensis, C. tropicalis, C. parapsilosis, C. krusei,and C. glabrata, and (2) AP-65 gene region of T. vaginalis. In someembodiments, the sample is contacted with the primers that canspecifically hybridize and amplify 16S rRNA genes of Atopobium vaginae,BVAB-2, Megasphaera Type 1, and vly gene of G. vaginalis, and the probesthat can specifically hybridizes to 16S rRNA genes of Atopobium vaginae,BVAB-2, Megasphaera Type 1, and vly gene of G. vaginalis.

The oligonucleotide probe can be, for example, between about 10 andabout 45 nucleotides in length, and comprises a detectable moiety. Insome embodiments, the contacting is performed under conditions allowingfor the specific hybridization of the primers to the correspondingtargeted gene region if the target organism is present in the sample.The presence and/or amount of probe that is specifically bound to thecorresponding targeted gene region (if present in the sample beingtested) can be determined, wherein bound probe is indicative of thepresence of the corresponding target organism in the sample. In someembodiments, the amount of bound probe is used to determine the amountof the corresponding target organism in the sample.

The determining step can be achieved using any methods known to thoseskilled in the art, including but not limited to, in situ hybridization,following the contacting step. The detection of hybrid duplexes (i.e.,of a probe specifically bound to the targeted gene region) can becarried out by a number of methods. Typically, hybridization duplexesare separated from unhybridized nucleic acids and the labels bound tothe duplexes are then detected. Such labels refer to radioactive,fluorescent, biological or enzymatic tags or labels of standard use inthe art. A label can be conjugated to either the oligonucleotide probesor the nucleic acids derived from the biological sample. Those of skillin the art will appreciate that wash steps may be employed to wash awayexcess sample/target nucleic acids or oligonucleotide probe (as well asunbound conjugate, where applicable). Further, standard heterogeneousassay formats are suitable for detecting the hybrids using the labelspresent on the oligonucleotide primers and probes.

Some embodiments provide a method to detect a plurality of BV-relatedbacteria in a biological sample, wherein the method comprises:contacting the biological sample with a plurality of pairs of primers,wherein the plurality of pairs of primer comprises: primers capable ofhybridizing to the 16S rRNA genes of L. crispatus and L. jensenii,wherein each primer comprises a sequence of SEQ ID NO: 14 or SEQ ID NO:15, or a sequence that exhibits at least about 85% identity to SEQ IDNO: 14 or SEQ ID NO: 15; primers capable of hybridizing to the 16S rRNAgene of BVAB2, wherein each primer comprises a sequence of SEQ ID NO: 4or SEQ ID NO: 5 or a sequence that exhibits at least about 85% identityto SEQ ID NO: 4 or SEQ ID NO: 5; primers capable of hybridizing to the16S rRNA gene of Megasphaera type 1, wherein each primer comprises asequence of SEQ ID NO: 7 or SEQ ID NO: 8 or a sequence that exhibits atleast about 85% identity to SEQ ID NO: 7 or SEQ ID NO: 8; primerscapable of hybridizing to the vly gene of G. vaginalis, wherein eachprimer comprises a sequence selected from the group consisting of SEQ IDNOS: 10-12 or a sequence that exhibits at least about 85% identity to asequence selected from the group consisting of SEQ ID NOS: 10-12, andprimers capable of hybridizing to the 16S rRNA gene of Atopobiumvaginae, wherein each primer comprises a sequence of SEQ ID NO: 1 or SEQID NO: 2, or sequence that exhibits at least about 85% identity to SEQID NO: 1 or SEQ ID NO: 2; generating amplicons of the 16S rRNA sequencesof Atopobium vaginae, BVAB2, Megasphaera type 1, and/or L. crispatus andL. jensenii, and/or amplicons of the vly gene sequence of G. vaginalisfrom said biological sample, if said sample comprises one or more of theBV-related bacteria; and determining the presence or amount of one ormore amplified products as an indication of the presence of BV-relatedbacteria in said biological sample.

In some embodiments, the plurality of pairs of primers comprises aprimer comprising the sequence of SEQ ID NO: 1, a primer comprising thesequence of SEQ ID NO: 2, a primer comprising the sequence of SEQ ID NO:4, a primer comprising the sequence of SEQ ID NO: 5, a primer comprisingthe sequence of SEQ ID NO: 7, a primer comprising the sequence of SEQ IDNO: 8, a primer comprising the sequence of SEQ ID NO: 10, a primercomprising the sequence of SEQ ID NO: 11, a primer comprising thesequence of SEQ ID NO: 12, a primer comprising the sequence of SEQ IDNO: 14, and a primer comprising the sequence of SEQ ID NO: 15. In someembodiments, the primers capable of hybridizing to the 16S rRNA genes ofLactobacillus crispatus and Lactobacillus jensenii comprise SEQ ID NOs:1 and 2; the primers capable of hybridizing to the 16S rRNA gene ofBVAB2 comprise SEQ ID NOs: 4 and 5; the primers capable of hybridizingto the 16S rRNA gene of Megasphaera type 1 comprise SEQ ID NOs: 7 and 8;the primers capable of hybridizing to the vly gene of G. vaginaliscomprise: (a) SEQ ID NOs: 10 and 12, (b) SEQ ID NOs: 11 and 12, or acombination thereof, and the primers capable of hybridizing to the 16SrRNA gene of Atopobium vaginae comprises SEQ ID NOs: 1 and 2.

In some embodiments, determining the presence or amount of one or moreamplified products comprises contacting the amplified products with aplurality of oligonucleotide probes, wherein each of the plurality ofoligonucleotide probes comprises a sequence selected from the groupconsisting of SEQ ID NOs: 3, 6, 9, 13, and 16, or a sequence thatexhibits at least about 85% identity to a sequence selected from thegroup consisting of SEQ ID NOs: 3, 6, 9, 13, and 16. For example, eachprobe can comprise, or consists of, a sequence selected from the groupconsisting of SEQ ID NOs: 3, 6, 9, 13, and 16.

Also disclosed herein is a method to detect VVC-associated Candidaspecies and T. vaginalis in a biological sample, wherein the methodcomprises: contacting the biological sample with a plurality of pairs ofprimers, wherein the plurality of pairs of primer comprises: primerscapable of hybridizing to the tef1 gene of C. glabrata, wherein eachprimer comprises a sequence of SEQ ID NO: 20 or SEQ ID NO: 21 or asequence that exhibits at least about 85% identity to SEQ ID NO: 20 orSEQ ID NO: 21; primers capable of hybridizing to the tef1 gene of atleast one of C. albicans, C. tropicalis, C. dubliniensis, and C.parapsilosis, wherein each primer comprises a sequence of SEQ ID NO: 23,SEQ ID NO: 24, or SEQ ID NO: 25, or a sequence that exhibits at leastabout 85% identity to SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 25;primers capable of hybridizing to the tef1 gene of C. krusei, whereineach primer comprises a sequence of SEQ ID NO: 27 or SEQ ID NO: 28, orsequence that exhibits at least about 85% identity to SEQ ID NO: 27 orSEQ ID NO: 28; and primers capable of hybridizing to the AP-65 gene ofT. vaginalis, wherein each primer comprises a sequence of SEQ ID NO: 17or SEQ ID NO: 18, or sequence that exhibits at least about 85% identityto SEQ ID NO: 17 or SEQ ID NO: 18; and generating amplicons of the tef1sequences of the Candida species and/or amplicons of the AP-65 genesequence of T. vaginalis from said biological sample, if said samplecomprises one or more of the VVC-associated Candida species and/or T.vaginalis; determining the presence or amount of one or more amplifiedproducts as an indication of the presence of VVC-associated Candidaspecies and T. vaginalis in said biological sample.

In some embodiments, the plurality of pairs of primers comprises aprimer comprising the sequence of SEQ ID NO: 20, a primer comprising thesequence of SEQ ID NO: 21, a primer comprising the sequence of SEQ IDNO: 23, a primer comprising the sequence of SEQ ID NO: 24, a primercomprising the sequence of SEQ ID NO: 25, a primer comprising thesequence of SEQ ID NO: 27, a primer comprising the sequence of SEQ IDNO: 28, a primer comprising the sequence of SEQ ID NO: 17, and a primercomprising the sequence of SEQ ID NO: 18.

In some embodiments, the primers capable of hybridizing to the tef1 geneof C. glabrata comprise SEQ ID NOs: 20 and 21; the primers capable ofhybridizing to the tef1 gene of at least one of C. albicans, C.tropicalis, C. dubliniensis, and C. parapsilosis comprise: (a) SEQ IDNOs: 23 and 24, (b) SEQ ID NOs: 23 and 35, or (c) a combination thereof,the primers capable of hybridizing to the tef1 gene of C. kruseicomprise of SEQ ID NOs: 27 and 28; and the primers capable ofhybridizing to the 16S rRNA gene of T. vaginalis comprise SEQ ID NOs: 17and 18.

In some embodiments, determining the presence or amount of one or moreamplified products comprises contacting the amplified products with aplurality of oligonucleotide probes, wherein each of the plurality ofoligonucleotide probes comprises a sequence selected from the groupconsisting of SEQ ID NOs: 3, 6, 9, 13, and 16, or a sequence thatexhibits at least about 85% identity to a sequence selected from thegroup consisting of SEQ ID NOs: 3, 6, 9, 13, and 16. In someembodiments, each of the plurality of oligonucleotide probes comprises,or consists of, a sequence selected from the group consisting of SEQ IDNOs: 3, 6, 9, 13, and 16.

As described herein, the amplification can be carried out by real-timePCR, for example, quantitative real-time PCR (QRT-PCR). The primerssuitable for use in the methods and compositions described herein cancomprise exogenous nucleotide sequence which allows post-amplificationmanipulation of amplification products without a significant effect onamplification itself. In some embodiments, the primer can be flanked bycomplementary sequences comprising a fluorophore at the 5′ end, and afluorescence quencher at the 3′ end.

The oligonucleotide probes disclosed herein can comprise a fluorescenceemitter moiety and a fluorescence quencher moiety

The methods disclosed herein are amendable to automation, therebyproviding a high-throughput option for the detection and/orquantification of VVC-associated Candida species, T. vaginalis, andBV-related bacteria in a sample. Various multiplex PCR platforms, e.g.,BD MAX™, Viper™, or Viper™ LT platforms, can be used to perform one ormore steps of the disclosed methods. The methods can be performed in amultiplex fashion. For example, the nucleic acid amplification and/ordetection, in some embodiments, comprise performing multiplex PCR.

EXAMPLES

The following examples are provided to demonstrate particular situationsand settings in which this technology may be applied and are notintended to restrict the scope of the invention and the claims includedin this disclosure.

Example 1 Detection of VVC, Trichomoniasis and BV in Vaginal SwabSamples

The study described in this example shows the detection of Candidaspecies associated with VVC, trichomoniasis and BV using an automatedqualitative in vitro diagnostic test in vaginal swab samples. The testutilizes real time PCR for the amplification of DNA targets andfluorogenic hybridization probes for the detection and identification oftarget organisms.

Vaginal swabs were collected from women with clinical symptoms ofvaginitis/vaginosis. Vaginal specimens were characterized by In Pouch™TV for T. vaginalis while culture followed by BD Phoenix™ identificationwas used for Candida species and the Nugent score (Nugen et al., J.Clin. Microbiol. 29(2):297-301 (1991)), as reference method for BV.Amsel's criteria (Amsel et al., Am. J. Med. 74(1):14-22 (1983)) wereused only in determination of BV statuses for specimens withintermediate Nugent score (Nugent's score 4-6). Three swabs were fortest on the BD MAX™ System (Becton, Dickinson and Company, New Jersey)for detection of trichomoniasis, Candida species associated with VVC,and BV using a Receiver Operating Characteristic (ROC) curve analysis.The diagnosis of BV was determined using an algorithm based on PCRparameters for the detection of BV-related bacteria, includingLactobacillus species, G. vaginalis, Atopobium vaginae, BVAB-2, andMegasphaera-1.

Real-time PCR for the amplification of DNA targets was performed usingthe primers provided in Table 1 and fluorogenic hybridization probesprovided in Table 1 were used to detect Candida species associated withVVC, T. vaginalis, and BV-related bacteria L. crispatus, L. jensenii, G.vaginalis, Atopobium vaginae, Megasphaera Type 1, and BVAB2 in each ofthe vaginal swab samples.

An inclusivity study was performed with cultivable strains originatingfrom 12 countries. The inclusivity study analysis was based onpositive/negative status of each individual target according toestablished PCR parameter thresholds. As shown in Table 2, the assay iscapable of detecting a large diversity of strains belonging to speciesinvolved in VVC, trichomoniasis and BV. The level of detection ofspecific organisms in mixtures demonstrated a high level of analyticalsensitivity, indicating that clinicians can be able to obtain a clearidentification of the pathogen(s) involved in vaginal infection andselect the treatment using only one vaginal specimen.

TABLE 2 Inclusivity Study Microorganism (Load/swab) Strain^(a) OriginStatus Candida albicans ATCC 18804 Uruguay POS (5.4 × 10⁵ CFU/swab) ATCC36232 ND POS ATCC 60193 USA POS ATCC 32032 South Africa POS CCUG 44014Sweden POS Atopobium vaginae CCUG 43049 Sweden POS (1.1 × 10³ CFU/swab)CCUG 44156 Sweden POS CCUG 55226 Belgium POS CCUG 44258 Sweden POS CCUG48515 Sweden POS Trichomonas vaginalis ATCC 30001 ND POS (1.4 × 10³Cells/swab) ATCC 30092 USA POS ATCC 30185 USA POS ATCC 30184 USA POSATCC 30237 USA POS Candida glabrata ATCC 2001 ND POS (2.8 × 10⁴CFU/swab) ATCC 15545 ND POS ATCC 90876 Germany POS YST-192^(b) USA POSATCC MYA-276 Scotland POS Candida krusei ATCC 6258 Sri Lanka POS (3.4 ×10⁴ CFU/swab) ATCC 28870 Italy POS ATCC 32672 New Zealand POS ATCC 44507England POS YST-367^(b) USA POS Gardnerella vaginalis ATCC 14018 USA POS(3.4 × 10⁴ CFU/swab) ATCC 14019 ND POS CCUG 44111 Sweden POS CCUG 44159Sweden POS CCUG 60143 A Sweden POS ATCC 49145 ND POS CCUG 44280 SwedenPOS Lactobacillus crispatus ATCC 33820 ND POS (1.4 × 10⁴ CFU/swab) CCUG44073 Sweden POS CCUG 42898 ND POS ATCC 33197 ND POS ATCC 53545 ND POSLactobacillus jensenii ATCC 25258 ND POS (2.1 × 10³ CFU/swab) CCUG 44492South Africa POS CCUG 44003 Sweden POS CCUG 44122 Sweden POS CCUG 44495South Africa POS Candida parapsilosis ATCC 22019 Puerto Rico POS (5.4 ×10⁵ CFU/swab) ATCC 28475 Norway POS YST-100^(b) Germany POS CCUG 37233Sweden POS YST-194 USA POS Candida tropicalis ATCC 750 ND POS (5.4 × 10⁵CFU/swab) ATCC 1369 ND POS ATCC 9968 former USSR POS YST-1051^(b) USAPOS CCUG 21298 Sweden POS ^(a)One strain from each microorganism testedin each mix ^(b)Strain from BD collection ND: not determined

In simulated co-infection studies, low load of T. vaginalis or a lowload of C. glabrata and C. krusei were tested in presence of high loadsof C. albicans; and low load of T. vaginalis was tested in presence of ahigh load of C. glabrata. For each study above, simulated matrix wasused rather than vaginal matrix due to the presence of some targets invaginal flora from asymptomatic/symptomatic women. The results of thesimulated co-infection studies are shown in Table 3.

TABLE 3 Stimulated co-infection study of the vaginal panel High load(Organisms/ Candida albicans Candida glabrata swab) (2.8E+6) (2.8E+6)Low load Candida Trichomonas Candida Trichomonas (Organisms/ kruseivaginalis glabrata vaginalis swab) (3.4E+4) (1.4E+3) (4.2E+3) (1.4E+3) %of conforming 95% 100% 100% 100% Assay results

Clinical specimens were defined as positive/negative sample for Candidaspecies and T. vaginalis. The results of the performance study shown inTable 4 demonstrate that the vaginal panel disclosed herein can be usedto detect T. vaginalis and Candida species with high sensitivity andspecificity.

TABLE 4 Performance study for TV and Candida species Vaginal Performancepanel Reference Sensitivity/ assay method Specificity^(a) Fraction^(b) %[2-sided 95 CI]^(e) T. vaginalis Inpouch Sensitivity 34/36 94.4[81.3-99.3] TV ™ Specificity 729/729 100.0 [99.6-100.0] Candida BDSensitivity 171/197 86.8 [81.3-91.2] species^(c, d) Phoenix ™Specificity 544/568 95.8 [93.8-97.3] ^(a)Sensitivity = True POS/TotalPOS from reference method and Specificity = True NEG/Total NEG fromreference method ^(b)«Unresolved» non-reportable results were excludedfrom Sensitivity/Specificity calculation «1 %) ^(c) Candida species: C.albicans, C. dubliniensis, C. guilliermondii, C. tropicalis, and C.parapsilosis ^(d) Candida glabrata and Candida krusei are detected intwo distinct channels: eight C. glabrata positive specimens for PCR andreference method, one positive for peR and two positive for referencemethod separately were obtained. One C. krusei positive specimen for PCRand reference method and one positive for each method separately wereobtained. ^(e)2-sided 95% Cl was calculated using the Clopper-Pearsonmethod

Assay performance for detection of BV was established using a ReceiverOperating Characteristic (ROC) curve analysis. Using PCR metrics fromthe amplification and detection of Lactobacillus species, g. vaginalis,Atopobium vaginae, BVAB-2 and Megasphaera-1, a logistic regressionmodel-based algorithm was built to estimate BV positive probability andgive a single BV positive or BV negative call. Patients were consideredto have BV if their estimated probability exceeds a threshold determinedby ROC curve analysis.

As shown in Table 5, preliminary assay performance results(sensitivity/specificity) based on analysis of 771 total characterizedclinical samples was 91.9% (sensitivity)/86.2% (specificity) orincreased to 95.4(sensitivity)/92.5% (specificity) when intermediateNugent Score and Amsel's criteria results were not considered.

TABLE 5 Performance study for BV detection Performance New BV ReferenceSensitivity/ Assay Method Specificity^(a) Fraction^(b) % [2-sided 95%CI] BV Nugent Sensitivity 350/381 91.9 [88.7-94.4] score/Amsel'sSpecificity 330/383 86.2 [82.3-89.5] criteria BV Nugent Sensitivity311/326^(c) 95.4 [92.5-97.4] score/Amsel's Specificity 297/321^(d) 92.5[89.1-95.2] criteria ^(a)Sensitivity = True POS/Total POS from referencemethod and Specificity = True NEG/Total NEG from reference method^(b)«Unresolved» non-reportable results were excluded fromSensitivity/Specificity calculation (<1%) ^(c)55 specimens withintermediate Nugent Score and classified as POS by Amsel's criteria wereexcluded. ^(d)62 specimens with intermediate Nugent Score and classifiedas NEG by Amsel's criteria were excluded.

This example demonstrates that the compositions and methods disclosedherein can be used to detect organisms related to VVC, TV, and BV withhigh specificity and sensitivity.

Example 2 Selection of Primers and Probes for Multiplex Detection ofVVC, Trichomoniasis and BV in Vaginal Samples

Various primers and probes have been designed and tested for theirperformance in amplification and detection of VVC-associated Candidaspecies, T. vaginalis, and BV individually or in a multiplex fashion.Table 6a and 6b provide various primers, primer pairs, and probes thatwere not selected because of a number of undesired properties, includingweak signal, lack of amplification, large size of amplicon, falsepositive signal, non-specific detection, sensitivity to temperaturevariation, failure to detect large number of variant strains,limitations in multiplex assay, selective of partner primers/probes,interaction with other primers/probes. Surprisingly, as described inExample 1, a number of primers and probes were found to perform well inthe amplification and detection of VVC-associated Candida species, T.vaginalis, and BV individually or in a multiplex fashion. The superiorproperties of those primers, probes and some combination thereof wereunpredicted. Moreover, the ability of the oligonucleotides of SEQ IDNOs: 1-16 to effectively perform (i.e., specifically amplify and detecttarget DNA) in a multiplex real-time PCR reaction was not predicted.Similarly, the ability of the oligonucleotides of SEQ ID NOs: 17-29 toeffectively perform (i.e., specifically amplify and detect target DNA)in a multiplex real-time PCR reaction also was not predicted.

TABLE 6a Non-selected primers and probes for detection of BV Analyte(target Targeted Non-selected primers, organism) geneprimer pairs and probes Primer and probe sequences (5′-3′) Atopobium16S rRNA HINAVFW GTTAGGTCAGGAGTTAAATCTG (SEQ ID NO: 33) vaginae HINAVRVTCATGGCCCAGAAGACC (SEQ ID NO: 34) HINAV-RVATCGTGGCCCAGAAGGCC (SEQ ID NO: 35) AVFP-BV1CCCTGGTAGTCCTAGCT (SEQ ID NO: 36) AVFP-BV1A*CCCTGGTAGTCCTAGCC (SEQ ID NO: 37) AVRP-BV1CGGCACGGAAAGTATAATCT (SEQ ID NO: 38) Forward primer (FW):GGTGAAGCAGTGGAAACACT (SEQ ID NO: 39) ATOVAGRT3FWGCAGCCCAGGACATAAGG (SEQ ID NO: 41) Reverse primer (RV):ATTCGCTTCTGCTCGCGCA (SEQ ID NO: 42) MCF-AV-R2 RV: ATOVAGRT3REV*FW: ATOP-442F GCAGGGACGAGGCCGCAA (SEQ ID NO: 43) RV: HINAVRVTCATGGCCCAGAAGACC (SEQ ID NO: 44) FW: MCF-AV-F1, andCGGATTCATTGGGCGTAAA (SEQ ID NO: 45) RV: MCF-AV-R3CGCCTCAGCGTCAGT (SEQ ID NO: 46) FW: MCF-AV-F1, andCGGATTCATTGGGCGTAAA (SEQ ID NO: 47) RV: MCF-AV-R4ACACCTAGTGTCCATCGTTTA (SEQ ID NO: 48) FW: MCF-AV-F2, andCCTTCGGGTTGTAAACCG (SEQ ID NO: 49) RV: MCF-AV-R3CGCCTCAGCGTCAGT (SEQ ID NO: 50) BVAB2 16S FW: HINBVAB2FW, andAGGCGGCTAGATAAGTGTGA (SEQ ID NO: 51) RV: HINBVAB2RVTCCTCTCCAGCACTCAAGCTAA (SEQ ID NO: 52) FW: BVAB2-619F, andTTAACCTTGGGGTTCATTACAA (SEQ ID NO: 53) RV: BVAB2-1024RAATTCAGTCTCCTGAATCGTCAGA (SEQ ID NO: 54) FW: BVAB2_585FAGCGGCTAGATAAGTGTGATGTTT (SEQ ID NO: 4) FW: HINBVAB2FWAGGCGGCTAGATAAGTGTGA (SEQ ID NO: 55) FW: BVABFP-BV2CGTGTAGGCGGCTAGATAAGTG (SEQ ID NO: 56) RV: BVAB2_879RGAATACTTATTGTGTTAACTGCGC (SEQ ID NO: 57) Megasphaera 16SFW: HINMGSTYP1FW, and GACGGATGCCAACAGTATCCGTCCG (SEQ ID NO: 7) type 1RV: HINMGSTYP1RV AAGTTCGACAGTTTCCGTCCCCTC (SEQ ID NO: 58) Gardnerellavaginolysin FW: GVVLYFW1, and GGCGGCGAAAGTGCTGTA (SEQ ID NO: 59)vaginalis (vly) RV: GVVLYRV1 AGCCGTTCACTGCGGAAGT (SEQ ID NO: 12)FW: GVVLYFW2, and GCCAACGATGATCGCGTAT (SEQ ID NO: 10) RV: GVVLYRV2ACAAGCTCGGCATGTTATCCAT (SEQ ID NO: 60) FW: MCF-GV-F6CCAGAATTTGATGGATAACATGCC (SEQ ID NO: 65) FW: MCF-GV-F7ATGGACAATATGCCAAGCCT (SEQ ID NO: 66) RV: MCF-GV-R2TTCACTGCGGAAGTTACAGA (SEQ ID NO: 67) RV: MCF-GV-R3TTAACTGCGGAAGTAACGGA (SEQ ID NO: 68) RV: MCF-GV-R4TTAACTGCTGAAGTAACGGA (SEQ ID NO: 69) Probes (5′ fluorophore:ACAGCACTTTCGCCGCC (SEQ ID NO: 13) Cy5; 3′ fluorophore:ACAGCACTCTCGCCGCC (SEQ ID NO: 70) BHQ2): MCF-GV-T3-CY5-B2MCF-GV-T4-CY5-B2 Gardnerella 16S rRNA FW: HINGVFW, andGGAAACGGGTGGTAATGCTGG (SEQ ID NO: 61) vaginalis RV: HINGVRVCGAAGCCTAGGTGGGCCATT (SEQ ID NO: 62) FW: GV1FW, andTTACTGGTGTATCACTGTAAGG (SEQ ID NO: 63) RV: GV3RVCCGTCACAGGCTGAACAGT (SEQ ID NO: 64) Lactobacillus 16S rRNAFW: L.crisp-452F, and GATAGAGGTAGTAACTGGCCTTTA (SEQ ID NO: 71) crispatusRV: L.crisp-1023R CTTTGTATCTCTACAAATGGCACTA (SEQ ID NO: 72)FW: HIN LG FW, and CGAGCTTGCCTAGATGAATTTG (SEQ ID NO: 73) RV: HIN LG RVCTCTAGACATGCGTCTAGTG (SEQ ID NO: 74) FW: HIN LC FW, andGATTTACTTCGGTAATGACGTTAGGA (SEQ ID NO: 75) RV: HIN LC RVAGCTGATCATGCGATCTGCTTTC (SEQ ID NO: 76) FW: HIN LJ FWGCCTATAGAAATTCTTCGGAATGGACA (SEQ ID NO: 77) RV: HIN LJ RVCAAATGGTATCCCAGACTTAAGGG (SEQ ID NO: 78) FW: MEG-LG_LJ-F6GTCGAGCGAGCTTGCCTA (SEQ ID NO: 79) FW: MCF-LC-F4GAACTAACAGATTTACTTCGGTAATG (SEQ ID NO: 80) RV: MCF-LG-R3AAACTCTAGACATGCGTCTAGT (SEQ ID NO: 81) RV: MCF-LJ_LC-R1GTTTCCAAATGGTATCCCAGA (SEQ ID NO: 82) Probes: Probes:MCF-Lj-Lc-T1_ROX-B2 CGGCGGATGGGTGAGTAAC (SEQ ID NO: 103)MCF-Lg-T5_ROX-B2 CCAAGAGACTGGGATAACACCTG (SEQ ID NO: 105)MCF-Lj-T7_ROX-B2 TCTTCGGAATGGACATAGATACAAGCTA (SEQ ID NO: 115)MCF-Lc-T3_ROX-B2 ATCCGCCGCTCGCTTT (SEQ ID NO: 116) FW: MCF-LG-F5GCCTAGATGAATTTGGTGCTT (SEQ ID NO: 83) FW: MCF-LJ-F6CGAGCTTGCCTATAGAAATTCTT (SEQ ID NO: 84) FW: MCF-LC-F4GAACTAACAGATTTACTTCGGTAATG (SEQ ID NO: 85) RV: MCF-LG-R3AAACTCTAGACATGCGTCTAGT (SEQ ID NO: 86) RV: MCF-LJ_LC-R1GTTTCCAAATGGTATCCCAGA (SEQ ID NO: 87) Probes: Probes:MCF-Lj-Lc-T1_ROX-B2 CGGCGGATGGGTGAGTAAC (SEQ ID NO: 103)MCF-Lg-T5_ROX-B2 CCAAGAGACTGGGATAACACCTG (SEQ ID NO: 105)FW: MCF-LJ_LC-F8 TTAAAAGGCGGCGTAAGC (SEQ ID NO: 14) FW: MCF-LG-F9ACTAGACGCATGTCTAGAGTTT (SEQ ID NO: 88) RV: MCF-LSP-R6GCCAGTTACTACCTCTATC (SEQ ID NO: 15)TGCATTAGCTAGTTGGTAAGGTAAC (SEQ ID NO: 89) Primers: Primers: MCF-Lj_Lc-F8TTAAAAGGCGGCGTAAGC (SEQ ID NO: 14) MCF-Lg-F9ACTAGACGCATGTCTAGAGTTT (SEQ ID NO: 88) MCF-Lj_Lc-R7GCCAGTTACTACCTCTATCCT (SEQ ID NO: 15) Probes: (5′ fluorophore: Probes:ROX; 3′ fluorophore: AAGTCTGATGGAGCAACGCC (SEQ ID NO: 16) BHQ2):ACATTGGGACTGAGACACGG (SEQ ID NO: 90) MCF-LSP-T8_ROX-B2AGGCTTACCAAGGCGATGAT (SEQ ID NO: 91) MCF-LSP-T11_ROX-B2CGGCTTACCAAGGCAATGAT (SEQ ID NO: 92) MCF-LSP-T13_ROX-B2MCF-LJ_LC-T15_ROX-B2 MCF-LG-T16_ROX-B2 FW: HIN LG FWCGAGCTTGCCTAGATGAATTTG (SEQ ID NO: 97) FW: HIN LJ FWGCCTATAGAAATTCTTCGGAATGGACA (SEQ ID NO: 98) FW: HIN LC FWGATTTACTTCGGTAATGACGTTAGGA (SEQ ID NO: 99) RV: HIN LG RVCTCTAGACATGCGTCTAGTG (SEQ ID NO: 100) RV: HIN LJ RVCAAATGGTATCCCAGACTTAAGGG (SEQ ID NO: 101) RV: HIN LC RVAGCTGATCATGCGATCTGCTTTC (SEQ ID NO: 102) Probes (5' fluorophore: Probes:ROX; 3' fluorophore: CGGCGGATGGGTGAGTAAC (SEQ ID NO: 103) BHQ2):CCAAGAGACTGGGATAACACCTG (SEQ ID NO: 105) MCF-Lj-Lc-T1_ROX-B2MCF-Lg-T5_ROX-B2

TABLE 6bNon-selected primers and probes for detection of VVC and trichomoniasisAnalyte (target Non-selected Primer and probe organism) Targeted geneprimers and probes sequences (5′-3′) Candida RNase P FW: cand-CR1CGGGTGGGAAATTCGGT (SEQ ID NO: 117) albicans RNA 1 RV: cand-CR5CAATGATCGGTATCGGGT (SEQ ID NO: 118) (RPR1) Probes: Probes: alb-T-FAM-B1CAGCTTGTAGTAAAGAATTACTCAC (SEQ ID NO: 119) cand-T-FAM-B1TTCGCATATTGCACTAAATAG (SEQ ID NO: 120) cand-Ta-FAM-B1TTCGCATATTGCACTAAACAG (SEQ ID NO: 121) Candida TopoisomeraseFW: MenCa1377fw CAACGCCAACGAAGACAAG (SEQ ID NO: 122) albicans IIIRV: MenCa1472rv CCAGCTTTGTTTGCATCAA (SEQ ID NO: 123) Probe: Probe:MenCa-T-FAM-B1 AAAGCCGATGGTAGTAGAAAACTGC (SEQ ID NO: 124) CandidaTopoisomerase FW: CABF59 TTGAACATCTCCAGTTTCAAAGGT (SEQ ID NO: 125)albicans II RV: CABR110 GTTGGCGTTGGCAATAGCTCTG (SEQ ID NO: 126) CandidaCHS1 FW: Jorprimer1Fw CGCCTCTTGATGGTGATGAT (SEQ ID NO: 127) speciesRV: Jorprimer2Rv TCCGGTATCACCTGGCTC (SEQ ID NO: 128) Probes: Probes:JorCa-T-FAM-B1 CGTTCGTACTAGAGTTGTGTTGTTTTGGAT (SEQ ID NO: 129)JorCpara-T-FAM-B1 GAGGCTGTGATGTGTGCTGTTGACCAG (SEQ ID NO: 130)JorCtro-T-FAM-B1 AGGCTTGCTCTTTGTCGGGCGAGCGAACG (SEQ ID NO: 131) CandidaTEF FW Primers: FW Primers: species ECanG278CAGGTCACAGAGATTTCATCAAG (SEQ ID NO: 132) cand-CR1-NP-CaGAAATTCGGTGGTACGCTCC (SEQ ID NO: 133) cand-CR1-NP-CtCpGAAATTCGGTGGTACTCTCC (SEQ ID NO: 134) RT-Ca_Cd-F2GTTGTGACTCTTTCAATGCCCAA (SEQ ID NO: 135) RT-Ctro-F3GTTGTGACTCTTTCAACGCTCAA (SEQ ID NO: 136) RT-Cpara-F4GATGTGACTCCTTCAATGCTCAA (SEQ ID NO: 137) RV Primers: RV Primers:ECanG401 GTAAGCCAACAAAGCGTGTTCTC (SEQ ID NO: 138) ECanG401aGAAAGCCAATAGAGCGTGTTCTC (SEQ ID NO: 139) Cand-CR5-NP-CaCtGATCGGTATCGGGTGCTTG (SEQ ID NO: 140) Cand-CR5-NP-CpGATCGGTATCGGGTTCTTG (SEQ ID NO: 141) RT-Cdub-R4CAGCGTCACCGGATTTGAC (SEQ ID NO: 142) Probes: Probes: ECanG-TL1-O2-FAM-B1TGATTATTGCTGGTGG (SEQ ID NO: 143) cand-T-FAM-B1TTCGCATATTGCACTAAATAG (SEQ ID NO: 120) cand-Ta-FAM-B1TTCGCATATTGCACTAAACAG (SEQ ID NO: 121) RT-Ca_Cd_Cp-T1-FAM-B1TGCTTGTAAATTCGACACTTTG (SEQ ID NO: 144) RT-Ctro-T4-FAM-B1TGTAAATTCGACACCTTGGTTGA (SEQ ID NO: 145) RT-Ca_Cd-T2-FAM-B1TTGTAAATTCGACACTTTGGTTG (SEQ ID NO: 146) RT-Cpar-T6-FAM-B1CGACACTTTGATTGAAAAGATTGAC (SEQ ID NO: 147) Candida ITS2 FW Primers:FW Primers: species ITS2-Ca-Fow GGGTTTGCTTGAAAGACGGTA (SEQ ID NO: 148)ITS2-Ctr-Fow CGTGGTAACTTATTTTAAGCG (SEQ ID NO: 149) ITS2-Cpar-FowGGGTTTGGTGTTGAGCGATAC (SEQ ID NO: 150) RV Primers: RV Primers:ITS2-Ca-Rev TTGAAGATATACGTGGTGGACGTTA (SEQ ID NO: 151) ITS2-Ctr-RevGCTTAAGTTCAGCGGGTAGTCCTA (SEQ ID NO: 152) ITS2-Cpar-RevGGAGTTTGTACCAATGAGTGGAAA (SEQ ID NO: 153) Probes: Probes ITS2-Ca-CFO-B1ACCTAAGCCATTGTCAAAGCGATCCCG (SEQ ID NO: 154) ITS2-Ctr-CFO-B1TGGCCACCATTTATTTCATAACTTTGACC (SEQ ID NO: 155) ITS2-Cpar-CFO-B1CTCCGCCTTTCTTTCAAGCAAACCCAG (SEQ ID NO: 156) Candida RNase P FW Primers:FW Primers: glabrata RNA 1 gla-CR3 GGCAACGGCTGGGAAT (SEQ ID NO: 157)(RPR1) gla-CR3a AGCAACGGCTGGGAAT (SEQ ID NO: 158) RV Primer: RV Primer:cand-CR5 CAATGATCGGTATCGGGT (SEQ ID NO: 159) Probe: Probe: gla-T-FAM-B1TAAAGCCTCACCACGATTTTGACAC (SEQ ID NO: 160) Candida TopoisomeraseFW Primer: CGBF35 CCCAAAAATGGCCGTAAGTATG (SEQ ID NO: 161) glabrata IIRV Primer: CGBR77 CTGCTTGAAAGAAATATCGGAGAC (SEQ ID NO: 162) Candida CHS1FW: Jorprimer1Fw CGCCTCTTGATGGTGATGAT (SEQ ID NO: 127) glabrataRV: Jorprimer2Rv TCCGGTATCACCTGGCTC (SEQ ID NO: 128) Probe: Probe:JorCgla-T-FAM-B1 CGACTGGTTGACGATAATCAGAGGAGATGGG (SEQ ID NO: 163)Candida TEF FW Primers: Primers: glabrata RT-Cgla-F5ACCCACCAAAGGCTGCT (SEQ ID NO: 164) RT-Cgla-F6CGACCCACCAAAGGCTGCT (SEQ ID NO: 165) Probe: Probe: RT-Cgla-T8-FAM-B1ACTGTCACACCGCCCACATT (SEQ ID NO: 166) Candida RNase PFW Primers: cand-CR1 CGGGTGGGAAATTCGGT (SEQ ID NO: 117) krusei RNA 1kru-CR1-SiT ATAGAGTAGCTCGGTCCC (SEQ ID NO: 167) (RPR1)RV Primers: krus-CR5 TAGTGATCGGTATCGAGTT (SEQ ID NO: 168) Kru-CR5-NP2CGGTATCGAGTTTCCATG (SEQ ID NO: 169) Probe: Probe: krus-T-FAM-B1CCAAAGTTGTACAAGCAAGTACCA (SEQ ID NO: 170) Candida TopoisomeraseFW: CKSF35 GAGCCACGGTAAAGAATACACA (SEQ ID NO: 171) kruseiII (K_(ANBE), 2002) RV: CKSR57 TTTAAAGTGACCCGGATACC (SEQ ID NO: 172)Candida TEF RV Primers: RT-Ckru-R5CTTTGGATGGTCTTCAACAGA (SEQ ID NO: 173) krusei SiT-Ckru-R10ATCACCAGACTTGACGG (SEQ ID NO: 174) Probes: Probes: SiT-Ckru-T10-CFO-B1AGTCTGTTGAAGACCATCCA (SEQ ID NO: 175) SiT-Ckru-T9-CFO-B1ATGTAAGTTCGACGAATTAATC (SEQ ID NO: 176) TV Ap65-1FW Primers: NP.TV.MAX.FP1 TCTGGCAAGATCAAGGACAT (SEQ ID NO: 177)SiT.TV.MAX.FP1 GAAGATTCTGGCAAGATCA (SEQ ID NO: 178)RV Primers: NP.TV.MAX.RP1 CATCTGTAACGACAATGCAGC (SEQ ID NO: 179)SiT.TV.MAX.RP1 GACAATGCAGCGGAT (SEQ ID NO: 180) Probes: Probes:NP.TV.MAX.D1-T-FAM-B1 AACTACCCACGCCAGGACAT (SEQ ID NO: 181)SiT.TV.MAX.D1-T-FAM-B1 CCGCAACTACCCACGCCA (SEQ ID NO: 182)

Tables 7a and 7b provide a number of master mixes of primers and probesthat were not selected because of a number of undesired properties,including false positive signal and failure to detect variant strains.

TABLE 7a Non-selected master mixes for detection of BV Master Mix IDPrimers and Probes Primer and Probe Sequences (5′-3′) Master Mix IPrimers: GCGGCTAGATAAGTGTGATGTTT (SEQ ID NO: 4) BVAB2_585FaCTCTCCAGCACTCAAGCTAAA (SEQ ID NO: 5) BVAB2_666RATTAAAAGGCGGCGTAAGC (SEQ ID NO: 14) MCF-LJ_LC-F8ACTAGACGCATGTCTAGAGTTT (SEQ ID NO: 88) MCF-LG-F9GCCAGTTACTACCTCTATC (SEQ ID NO: 15) MCF-LSP-R6CCCTATCCGCTCCTGATACC (SEQ ID NO: 1) MENAV248FWCCAAATATCTGCGCATTTCA (SEQ ID NO: 2) MENAV334RVCCAGAATTTGATGGATAACATGCC (SEQ ID NO: 65) MCF-GV-F6ATGGACAATATGCCAAGCCT (SEQ ID NO: 66) MCF-GV-F7TTCACTGCGGAAGTTACAGA (SEQ ID NO: 67) MCF-GV-R2TTAACTGCTGAAGTAACGGA (SEQ ID NO: 69) MCF-GV-R4GATGCCAACAGTATCCGTCCG (SEQ ID NO: 7) MEGAE-456FCCTCTCCGACACTCAAGTTCGA (SEQ ID NO: 8) MEGAE-667R Probes:FAM-CAAGGCTTAACCTTGGGGTTCATTACAA-BHQ1 (SEQ ID NO: 6)BVAB2_613_641_FAM-B1 ROX-AAGTCTGATGGAGCAACGCC-BHQ2 (SEQ ID NO: 16)MCF-LSP-T11_ROX-B2 FAM-TCCCCTACCAGACTCAAGCCTGC-BHQ1 (SEQ ID NO: 3)MCF-AV-T4_FAM-B1 Cy5-ACAGCACTTTCGCCGCC-BHQ2 (SEQ ID NO: 13)MCF-GV-T3-CY5-B2 Cy5-ACAGCACTCTCGCCGCC-BHQ2 (SEQ ID NO: 70)MCF-GV-T4-CY5-B2 HEX-GTACCGTAAGAGAAAGCCACGG-BHQ1 (SEQ ID NO: 9)MEGA_485-506-T-HEX-BHQ1 Master Mix II Primers:GCGGCTAGATAAGTGTGATGTTT (SEQ ID NO: 4) BVAB2_585FaCTCTCCAGCACTCAAGCTAAA (SEQ ID NO: 5) BVAB2_666RaTTAAAAGGCGGCGTAAGC (SEQ ID NO: 14) MCF-Lj_Lc-F8ACTAGACGCATGTCTAGAGTTT (SEQ ID NO: 88) MCF-Lg-F9GCCAGTTACTACCTCTATC (SEQ ID NO: 15) MCF-Lsp-R6CCCTATCCGCTCCTGATACC (SEQ ID NO: 1) MenAv248fwCCAAATATCTGCGCATTTCA (SEQ ID NO: 2) MenAv334rvCGCATCTGCTAAGGATGTTG (SEQ ID NO: 106) MenGV981fw,CAGCAATCTTTTCGCCAACT (SEQ ID NO: 107) MenGV1072rvGATGCCAACAGTATCCGTCCG (SEQ ID NO: 7) MegaE-456FCCTCTCCGACACTCAAGTTCGA (SEQ ID NO: 8) MegaE-667R Probes:FAM-CAAGGCTTAACCTTGGGGTTCATTACAA-BHQ1 (SEQ ID NO: 6)BVAB2_613_641_CFO-B1 ROX-AAGTCTGATGGAGCAACGCC-BHQ2 (SEQ ID NO: 16)MCF-Lsp-T11_ROX-B2 FAM-TCCCCTACCAGACTCAAGCCTGC-BHQ1 (SEQ ID NO: 3)MCF-Av-T4_FAM-B1 ROX-TGCAACTATTTCTGCAGCAGATCC-BHQ2 (SEQ ID NO: 108)MenGV-T-ROX-B2 CFO-GTACCGTAAGAGAAAGCCACGG-BHQ1 (SEQ ID NO: 9)Mega_485-506-T-CFO-BHQ1 Master Mix IIII Primers:GCGGCTAGATAAGTGTGATGTTT (SEQ ID NO: 4) BVAB2_585FaCTCTCCAGCACTCAAGCTAAA (SEQ ID NO: 5) BVAB2_666RaTTAAAAGGCGGCGTAAGC (SEQ ID NO: 14) MCF-Lj_Lc-F8ACTAGACGCATGTCTAGAGTTT (SEQ ID NO: 88) MCF-Lg-F9GCCAGTTACTACCTCTATC (SEQ ID NO: 15) MCF-Lsp-R6CCCTATCCGCTCCTGATACC (SEQ ID NO: 1) MenAv248fwCCAAATATCTGCGCATTTCA (SEQ ID NO: 2) MenAv334rvGCCAACGATGATCGCGTAT (SEQ ID NO: 10) GVvlyfw2CAGGCTTGGCATATTGTCCAT (SEQ ID NO: 109) GVvlyrv2GCCAATAATGACCGCGTAT (SEQ ID NO: 11) GVvlyfw2aCAAGCTCGGCATGTTATCCAT (SEQ ID NO: 60) GVvlyrv2aGATGCCAACAGTATCCGTCCG (SEQ ID NO: 7) MegaE-456FCCTCTCCGACACTCAAGTTCGA (SEQ ID NO: 8) MegaE-667R Probes:FAM-CAAGGCTTAACCTTGGGGTTCATTACAA-BHQ1 (SEQ ID NO: 6)BVAB2_613_641_CFO-B1 ROX-AAGTCTGATGGAGCAACGCC-BHQ2 (SEQ ID NO: 16)MCF-Lsp-T11_ROX-B2 FAM-TCCCCTACCAGACTCAAGCCTGC-BHQ1 (SEQ ID NO: 3)MCF-Av-T4_FAM-B1 ROX-CCCAGGTGCTCTTTTCCGTGCTGA-BHQ2 (SEQ ID NO: 110)GVvly-T2-ROX-B2 ROX-CCCAGGTGCGCTGTTCCGCGCTGA-BHQ2 (SEQ ID NO: 111)GVvly-T2a-ROX-B2 CFO-GTACCGTAAGAGAAAGCCACGG-BHQ1 (SEQ ID NO: 9)Mega_485-506-T-CFO-BHQ1 Master Mix IV Primers:GCGGCTAGATAAGTGTGATGTTT (SEQ ID NO: 4) BVAB2_585FaCTCTCCAGCACTCAAGCTAAA (SEQ ID NO: 5) BVAB2_666RaTTAAAAGGCGGCGTAAGC (SEQ ID NO: 14) MCF-Lj_Lc-F8ACTAGACGCATGTCTAGAGTTT (SEQ ID NO: 88) MCF-Lg-F9GCCAGTTACTACCTCTATC (SEQ ID NO: 15) MCF-Lsp-R6CCCTATCCGCTCCTGATACC (SEQ ID NO: 1) MenAv248fwCCAAATATCTGCGCATTTCA (SEQ ID NO: 2) MenAv334rvGGCGGCGAAAGTGCTGTA (SEQ ID NO: 59) GVvlyfw1AGCCGTTCACTGCGGAAGT (SEQ ID NO: 12) GVvlyrv1GGCGGCGAAAGTGCTGTC (SEQ ID NO: 112) GVvlyfw1aGATGCCAACAGTATCCGTCCG (SEQ ID NO: 7) MegaE-456FCCTCTCCGACACTCAAGTTCGA (SEQ ID NO: 8) MegaE-667R Probes:FAM-CAAGGCTTAACCTTGGGGTTCATTACAA-BHQ1 (SEQ ID NO: 6)BVAB2_613_641_CFO-B1 ROX-AAGTCTGATGGAGCAACGCC-BHQ2 (SEQ ID NO: 16)MCF-Lsp-T11_ROX-B2 FAM-TCCCCTACCAGACTCAAGCCTGC-BHQ1 (SEQ ID NO: 3)MCF-Av-T4_FAM-B1 ROX-TTCAGCGCCCAACCAAGAGCTCTGT-BHQ2 (SEQ ID NO: 113)GVvly-T1-ROX-B2 ROX-TTAAGCATCCAACTAAGAGCTCTGT-BHQ2 (SEQ ID NO: 114)Gvvly-T1a-ROX-B2 CFO-GTACCGTAAGAGAAAGCCACGG-BHQ1 (SEQ ID NO: 9)Mega_485-506-T-CFO-BHQ1

TABLE 7bNon-selected master mixes for detection of VVC and trichomoniasisMaster Mix ID Primers and Probes Primer and Probe Sequences (5′-3′)Master Mix I SiT-Cgla-F8 CGAACAATTGACTGAAGGTTTG (SEQ ID NO: 20)RT-Cgla-R7 CGGACTTCAAGAACTTTGGAGA (SEQ ID NO: 21) RT-Cgla-T7-Fam-B1CTTGTAAGTTCGAAGAATTGTTGGA (SEQ ID NO: 22) TV.MAX.FP1GAAGATTCTGGCAAGATCAAGGA (SEQ ID NO: 17) TV.MAX.RP1ACGACAATGCAGCGGATGT (SEQ ID NO: 18) TV.MAX.D1-T-ROX-B2ATCCTCCGCAACTACCCACGCCA (SEQ ID NO: 19) kru-CR1-SiTATAGAGTAGCTCGGTCCC (SEQ ID NO: 167) Kru-CR5-NP2CGGTATCGAGTTTCCATG (SEQ ID NO: 169) krus-T-FAM-B1CCAAAGTTGTACAAGCAAGTACCA (SEQ ID NO: 170) RT-Ca_Cd_Ct-F1CCACCAAAGGGTTGTGAC (SEQ ID NO: 23) RT-Cpara-F4GATGTGACTCCTTCAATGCTCAA (SEQ ID NO: 137) RT-Ca_Ct-R3CAGCATCACCGGATTTGAC (SEQ ID NO: 24) RT-Cpar-R6CGGACTTGATGAATTTTGGTTCA (SEQ ID NO: 25) RT-Cdub-R4CAGCGTCACCGGATTTGAC (SEQ ID NO: 142) RT-Ca_Cd_Cp-T1-FAM-B1TGCTTGTAAATTCGACACTTTG (SEQ ID NO: 144) RT-Ctro-T4-FAM-B1TGTAAATTCGACACCTTGGTTGA (SEQ ID NO: 145) Master Mix II SiT-Cgla-F8CGAACAATTGACTGAAGGTTTG (SEQ ID NO: 20) RT-Cgla-R7CGGACTTCAAGAACTTTGGAGA (SEQ ID NO: 21) RT-Cgla-T7-Fam-B1CTTGTAAGTTCGAAGAATTGTTGGA (SEQ ID NO: 22) TV.MAX.FP1GAAGATTCTGGCAAGATCAAGGA (SEQ ID NO: 17) TV.MAX.RP1ACGACAATGCAGCGGATGT (SEQ ID NO: 18) TV.MAX.D1-T-ROX-B2ATCCTCCGCAACTACCCACGCCA (SEQ ID NO: 19) kru-CR1-SiTATAGAGTAGCTCGGTCCC (SEQ ID NO: 167) Kru-CR5-NP2CGGTATCGAGTTTCCATG (SEQ ID NO: 169) krus-T-FAM-B1CCAAAGTTGTACAAGCAAGTACCA (SEQ ID NO: 170) RT-Ca_Cd-F2GTTGTGACTCTTTCAATGCCCAA (SEQ ID NO: 135) RT-Ctro-F3GTTGTGACTCTTTCAACGCTCAA (SEQ ID NO: 136) RT-Cpara-F4GATGTGACTCCTTCAATGCTCAA (SEQ ID NO: 137) RT-Ca_Ct-R3CAGCATCACCGGATTTGAC (SEQ ID NO: 24) RT-Cpar-R6CGGACTTGATGAATTTTGGTTCA (SEQ ID NO: 25) RT-Cdub-R4CAGCGTCACCGGATTTGAC (SEQ ID NO: 142) RT-Ca_Cd-T3-FAM-B1TGCTTGTAAATTCGACACTTTGGTTG (SEQ ID NO: 26) RT-Ctro-T4-FAM-B1TGTAAATTCGACACCTTGGTTGA (SEQ ID NO: 145) RT-Cpar-T6-FAM-B1CGACACTTTGATTGAAAAGATTGAC (SEQ ID NO: 147) Master Mix III SiT-Cgla-F8CGAACAATTGACTGAAGGTTTG (SEQ ID NO: 20) RT-Cgla-R7CGGACTTCAAGAACTTTGGAGA (SEQ ID NO: 21) RT-Cgla-T7-Fam-B1CTTGTAAGTTCGAAGAATTGTTGGA (SEQ ID NO: 22) TV.MAX.FP1GAAGATTCTGGCAAGATCAAGGA (SEQ ID NO: 17) TV.MAX.RP1ACGACAATGCAGCGGATGT (SEQ ID NO: 18) TV.MAX.D1-T-ROX-B2ATCCTCCGCAACTACCCACGCCA (SEQ ID NO: 19) RT-Ckru-F7GCAGCTTCCTTCAATGCTCAA (SEQ ID NO: 27) RT-Ckru-R5CTTTGGATGGTCTTCAACAGA (SEQ ID NO: 173) RT-Ckru-T9-FAM-B1CATGTAAGTTCGACGAATTAATCGA (SEQ ID NO: 29) RT-Ca_Cd_Ct-F1CCACCAAAGGGTTGTGAC (SEQ ID NO: 23) RT-Ca_Ct-R3CAGCATCACCGGATTTGAC (SEQ ID NO: 24) RT-Cpar-R6CGGACTTGATGAATTTTGGTTCA (SEQ ID NO: 25) RT-Ca_Cd_Cp-T1-FAM-B1TGCTTGTAAATTCGACACTTTG (SEQ ID NO: 144) Master Mix IV SiT-Cgla-F8CGAACAATTGACTGAAGGTTTG (SEQ ID NO: 20) RT-Cgla-R7CGGACTTCAAGAACTTTGGAGA (SEQ ID NO: 21) RT-Cgla-T7-Fam-B1CTTGTAAGTTCGAAGAATTGTTGGA (SEQ ID NO: 22) TV.MAX.FP1GAAGATTCTGGCAAGATCAAGGA (SEQ ID NO: 17) TV.MAX.RP1ACGACAATGCAGCGGATGT (SEQ ID NO: 18) TV.MAX.D1-T-ROX-B2ATCCTCCGCAACTACCCACGCCA (SEQ ID NO: 19) RT-Ckru-F7GCAGCTTCCTTCAATGCTCAA (SEQ ID NO: 27) SiT-Ckru-R10ATCACCAGACTTGACGG (SEQ ID NO: 174) SiT-Ckru-T9-CFO-B1ATGTAAGTTCGACGAATTAATC (SEQ ID NO: 176) RT-Ca_Cd_Ct-F1CCACCAAAGGGTTGTGAC (SEQ ID NO: 23) RT-Ca_Ct-R3CAGCATCACCGGATTTGAC (SEQ ID NO: 24) RT-Ca_Cd_Cp-T1-FAM-B1TGCTTGTAAATTCGACACTTTG (SEQ ID NO: 144) Master Mix V SiT-Cgla-F8CGAACAATTGACTGAAGGTTTG (SEQ ID NO: 20) RT-Cgla-R7CGGACTTCAAGAACTTTGGAGA (SEQ ID NO: 21) RT-Cgla-T7-Fam-B1CTTGTAAGTTCGAAGAATTGTTGGA (SEQ ID NO: 22) TV.MAX.FP1GAAGATTCTGGCAAGATCAAGGA (SEQ ID NO: 17) TV.MAX.RP1ACGACAATGCAGCGGATGT (SEQ ID NO: 18) TV.MAX.D1-T-ROX-B2ATCCTCCGCAACTACCCACGCCA (SEQ ID NO: 19) RT-Ckru-F7GCAGCTTCCTTCAATGCTCAA (SEQ ID NO: 27) SiT-Ckru-R10aATCACCAGACTTGACAG (SEQ ID NO: 28) SiT-Ckru-T10-CFO-B1AGTCTGTTGAAGACCATCCA (SEQ ID NO: 175) RT-Ca_Cd_Ct-F1CCACCAAAGGGTTGTGAC (SEQ ID NO: 23) RT-Ca_Ct-R3CAGCATCACCGGATTTGAC (SEQ ID NO: 24) RT-Ca_Cd_Cp-T1-FAM-B1TGCTTGTAAATTCGACACTTTG (SEQ ID NO: 144)

While various aspects and embodiments have been disclosed herein, otheraspects and embodiments will be apparent to those skilled in the art.The various aspects and embodiments disclosed herein are for purposes ofillustration and are not intended to be limiting, with the true scopeand spirit being indicated by the following claims.

One skilled in the art will appreciate that, for this and otherprocesses and methods disclosed herein, the functions performed in theprocesses and methods can be implemented in differing order.Furthermore, the outlined steps and operations are only provided asexamples, and some of the steps and operations can be optional, combinedinto fewer steps and operations, or expanded into additional steps andoperations without detracting from the essence of the disclosedembodiments.

With respect to the use of substantially any plural and/or singularterms herein, those having skill in the art can translate from theplural to the singular and/or from the singular to the plural as isappropriate to the context and/or application. The varioussingular/plural permutations may be expressly set forth herein for sakeof clarity.

It will be understood by those within the art that, in general, termsused herein, and especially in the appended claims (e.g., bodies of theappended claims) are generally intended as “open” terms (e.g., the term“including” should be interpreted as “including but not limited to,” theterm “having” should be interpreted as “having at least,” the term“includes” should be interpreted as “includes but is not limited to,”etc.). It will be further understood by those within the art that if aspecific number of an introduced claim recitation is intended, such anintent will be explicitly recited in the claim, and in the absence ofsuch recitation no such intent is present. For example, as an aid tounderstanding, the following appended claims may contain usage of theintroductory phrases “at least one” and “one or more” to introduce claimrecitations. However, the use of such phrases should not be construed toimply that the introduction of a claim recitation by the indefinitearticles “a” or “an” limits any particular claim containing suchintroduced claim recitation to embodiments containing only one suchrecitation, even when the same claim includes the introductory phrases“one or more” or “at least one” and indefinite articles such as “a” or“an” (e.g., “a” and/or “an” should be interpreted to mean “at least one”or “one or more”); the same holds true for the use of definite articlesused to introduce claim recitations. In addition, even if a specificnumber of an introduced claim recitation is explicitly recited, thoseskilled in the art will recognize that such recitation should beinterpreted to mean at least the recited number (e.g., the barerecitation of “two recitations,” without other modifiers, means at leasttwo recitations, or two or more recitations). Furthermore, in thoseinstances where a convention analogous to “at least one of A, B, and C,etc.” is used, in general such a construction is intended in the senseone having skill in the art would understand the convention (e.g., “asystem having at least one of A, B, and C” would include but not belimited to systems that have A alone, B alone, C alone, A and Btogether, A and C together, B and C together, and/or A, B, and Ctogether, etc.). In those instances where a convention analogous to “atleast one of A, B, or C, etc.” is used, in general such a constructionis intended in the sense one having skill in the art would understandthe convention (e.g., “a system having at least one of A, B, or C” wouldinclude but not be limited to systems that have A alone, B alone, Calone, A and B together, A and C together, B and C together, and/or A,B, and C together, etc.). It will be further understood by those withinthe art that virtually any disjunctive word and/or phrase presenting twoor more alternative terms, whether in the description, claims, ordrawings, should be understood to contemplate the possibilities ofincluding one of the terms, either of the terms, or both terms. Forexample, the phrase “A or B” will be understood to include thepossibilities of “A” or “B” or “A and B.”

In addition, where features or aspects of the disclosure are describedin terms of Markush groups, those skilled in the art will recognize thatthe disclosure is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

As will be understood by one skilled in the art, for any and allpurposes, such as in terms of providing a written description, allranges disclosed herein also encompass any and all possible subrangesand combinations of subranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” and the like include the number recited andrefer to ranges which can be subsequently broken down into subranges asdiscussed above.

Whenever a range of values is provided herein, the range is meant toinclude the starting value, the ending value, each individual value, orvalue range there between unless otherwise specifically stated. Forexample, “from 0.2 to 0.5” means 0.2, 0.3, 0.4, 0.5; ranges therebetween such as 0.2-0.3, 0.3-0.4, 0.2-0.4; increments there between suchas 0.25, 0.35, 0.225, 0.335, 0.49; increment ranges there between suchas 0.26-0.39; and the like. As another example, a group having 1-3 cellsrefers to groups having 1, 2, or 3 cells. Similarly, a group having 1-5cells refers to groups having 1, 2, 3, 4, or 5 cells, and so forth.

From the foregoing, it will be appreciated that various embodiments ofthe present disclosure have been described herein for purposes ofillustration, and that various modifications may be made withoutdeparting from the scope and spirit of the present disclosure.Accordingly, the various embodiments disclosed herein are not intendedto be limiting, with the true scope and spirit being indicated by thefollowing claims.

What is claimed is:
 1. A method to detect vulvovaginal candidiasis(VVC)-associated Candida species and Trichomonas vaginalis in abiological sample, wherein the VVC-associated Candida species comprisesCandida glabrata, Candida albicans, Candida tropicalis, C. dubliniensis,C. parapsilosis, Candida krusei, comprising: contacting said biologicalsample with a plurality of pairs of primers, wherein the plurality ofpairs of primer comprises: at least one pair of primers capable ofhybridizing to the tef1 gene of Candida glabrata, wherein each primer insaid at least one pair of primers comprises a sequence of SEQ ID NO: 20or SEQ ID NO: 21 or a sequence that exhibits at least about 85% identityto SEQ ID NO: 20 or SEQ ID NO: 21; a plurality of primers capable ofhybridizing to the tef1 gene of at least one of Candida albicans,Candida tropicalis, C. dubliniensis, and C. parapsilosis, wherein eachprimer in said at least one pair of primers comprises a sequence of SEQID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 25, or a sequence that exhibitsat least about 85% identity to SEQ ID NO: 23, SEQ ID NO: 24, or SEQ IDNO: 25; at least one pair of primers capable of hybridizing to the tef1gene of Candida krusei, wherein each primer in said at least one pair ofprimers comprises a sequence of SEQ ID NO: 27 or SEQ ID NO: 28, orsequence that exhibits at least about 85% identity to SEQ ID NO: 27 orSEQ ID NO: 28; and at least one pair of primers capable of hybridizingto the AP-65 gene of Trichomonas vaginalis, wherein each primer in saidat least one pair of primers comprises a sequence of SEQ ID NO: 17 orSEQ ID NO: 18, or sequence that exhibits at least about 85% identity toSEQ ID NO: 17 or SEQ ID NO: 18; and generating amplicons of the tef1sequences of the Candida species and/or amplicons of the AP-65 genesequence of Trichomonas vaginalis from said biological sample, if saidsample comprises one or more of the VVC-associated Candida speciesand/or Trichomonas vaginalis; determining the presence or amount of oneor more amplified products as an indication of the presence ofVVC-associated Candida species and Trichomonas vaginalis in saidbiological sample.
 2. The method of claim 1, wherein said biologicalsample is a clinical sample.
 3. The method of claim 1, wherein saidbiological sample is collected from the urethra, penis, anus, throat,cervix, or vagina.
 4. The method of claim 1, wherein said biologicalsample is a vaginal sample.
 5. The method of claim 1, wherein theplurality of pairs of primers comprises a first primer comprising thesequence of SEQ ID NO: 20, a second primer comprising the sequence ofSEQ ID NO: 21, a third primer comprising the sequence of SEQ ID NO: 23,a fourth primer comprising the sequence of SEQ ID NO: 24, a fifth primercomprising the sequence of SEQ ID NO: 25, a sixth primer comprising thesequence of SEQ ID NO: 27, a seventh primer comprising the sequence ofSEQ ID NO: 28, an eighth primer comprising the sequence of SEQ ID NO:17, and a ninth primer comprising the sequence of SEQ ID NO:
 18. 6. Themethod of claim 1, wherein the pair of primers capable of hybridizing tothe tef1 gene of Candida glabrata is SEQ ID NOs: 20 and 21; the primerscapable of hybridizing to the tef1 gene of at least one of Candidaalbicans, Candida tropicalis, C. dubliniensis, and C. parapsilosis are:a) SEQ ID NOs: 23 and 24, b) SEQ ID NOs: 23 and 35, or c) a combinationthereof, the pair of primers capable of hybridizing to the tef1 gene ofCandida krusei consists of SEQ ID NOs: 27 and 28; and the pair ofprimers capable of hybridizing to the 16S rRNA gene of Trichomonasvaginalis is SEQ ID NOs: 17 and
 18. 7. The method of claim 1, whereinsaid amplification is carried out using a method selected from the groupconsisting of polymerase chain reaction (PCR), ligase chain reaction(LCR), loop-mediated isothermal amplification (LAMP), stranddisplacement amplification (SDA), replicase-mediated amplification,Immuno-amplification, nucleic acid sequence based amplification (NASBA),self-sustained sequence replication (3SR), rolling circle amplification,and transcription-mediated amplification (TMA).
 8. The method of claim7, wherein said PCR is real-time PCR.
 9. The method of claim 1, whereineach primer comprises exogenous nucleotide sequence which allowspost-amplification manipulation of amplification products without asignificant effect on amplification itself.
 10. The method of claim 1,wherein each primer is flanked by complementary sequences comprising afluorophore at the 5′ end, or a fluorescence quencher at the 3′ end, orboth.
 11. The method of claim 1, wherein determining the presence oramount of one or more amplified products comprises contacting theamplified products with a plurality of oligonucleotide probes, whereineach of the plurality of oligonucleotide probes comprises a sequenceselected from the group consisting of SEQ ID NOs: 3, 6, 9, 13, and 16,or a sequence that exhibits at least about 85% identity to a sequenceselected from the group consisting of SEQ ID NOs: 3, 6, 9, 13, and 16.12. The method of claim 11, wherein each of the plurality ofoligonucleotide probes comprises a sequence selected from the groupconsisting of SEQ ID NOs: 3, 6, 9, 13, and
 16. 13. The method of claim12, wherein each of the plurality of oligonucleotide probes consists ofa sequence selected from the group consisting of SEQ ID NOs: 3, 6, 9,13, and
 16. 14. The method of claim 11, wherein at least one of theplurality of oligonucleotide probes comprises a fluorescence emittermoiety, a fluorescence quencher moiety, or both.
 15. A composition forthe detection of vulvovaginal candidiasis (VVC)-associated Candidaspecies and Trichomonas vaginalis in a biological sample, wherein theVVC-associated Candida species comprises Candida glabrata, Candidaalbicans, Candida tropicalis, C. dubliniensis, C. parapsilosis, Candidakrusei, comprising: at least one pair of primers capable of hybridizingto the tef1 gene of Candida glabrata, wherein each primer in said atleast one pair of primers comprises a sequence of SEQ ID NO: 20 or SEQID NO: 21 or a sequence that exhibits at least about 85% identity to SEQID NO: 20 or SEQ ID NO: 21; a plurality of primers capable ofhybridizing to the tef1 gene of at least one of Candida albicans,Candida tropicalis, C. dubliniensis, and C. parapsilosis, wherein eachprimer in said at least one pair of primers comprises a sequence of SEQID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 25, or a sequence that exhibitsat least about 85% identity to SEQ ID NO: 23, SEQ ID NO: 24, or SEQ IDNO: 25; at least one pair of primers capable of hybridizing to the tef1gene of Candida krusei, wherein each primer in said at least one pair ofprimers comprises a sequence of SEQ ID NO: 27 or SEQ ID NO: 28, orsequence that exhibits at least about 85% identity to SEQ ID NO: 27 orSEQ ID NO: 28; and at least one pair of primers capable of hybridizingto the AP-65 gene of Trichomonas vaginalis, wherein each primer in saidat least one pair of primers comprises a sequence of SEQ ID NO: 17 orSEQ ID NO: 18, or sequence that exhibits at least about 85% identity toSEQ ID NO: 17 or SEQ ID NO:
 18. 16. The composition of claim 15, whereinthe at least one pair of primers capable of hybridizing to the tef1 geneof Candida glabrata comprises a primer comprising the sequence of SEQ IDNO: 20 and a primer comprising the sequence of SEQ ID NO: 21; theplurality of primers capable of hybridizing to the tef1 gene of at leastone of Candida albicans, Candida tropicalis, C. dubliniensis, and C.parapsilosis comprises a primer comprising the sequence of SEQ ID NO:23, a primer comprising the sequence of SEQ ID NO: 24, and a primercomprising the sequence of SEQ ID NO: 25; the at least one pair ofprimers capable of hybridizing to the tef1 gene of Candida kruseicomprises a primer comprising the sequence of SEQ ID NO: 27 and a primercomprising the sequence of SEQ ID NO: 28; and the at least one pair ofprimers capable of hybridizing to the AP-65 gene of Trichomonasvaginalis comprises a primer comprising the sequence of SEQ ID NO: 17and a primer comprising the sequence of SEQ ID NO:
 18. 17. Thecomposition of claim 15, further comprising a plurality ofoligonucleotide probes, wherein each of the plurality of oligonucleotideprobes comprises a sequence selected from the group consisting of SEQ IDNOs: 22, 26, 29, and 19, or a sequence that exhibits at least about 85%identity to a sequence selected from the group consisting of SEQ ID NOs:22, 26, 29, and
 19. 18. The composition of claim 17, wherein each of theplurality of oligonucleotide probes comprises a sequence selected fromthe group consisting of SEQ ID NOs: 22, 26, 29, and
 19. 19. Thecomposition of claim 18, wherein each of the plurality ofoligonucleotide probes consists of a sequence selected from the groupconsisting of SEQ ID NOs: 22, 26, 29, and
 19. 20. The composition ofclaim 17, wherein at least one of the plurality of probes comprises afluorescence emitter moiety, a fluorescence quencher moiety, or both.